Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.     

Archaeal replicative primases can perform translesion DNA synthesis

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2''-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.The DNA replication machinery rapidly and accurately copies genomes but is prone to stalling at lesions and physical barriers (1). A variety of cellular pathways have evolved to restart stalled replication forks. These include translesion DNA synthesis (TLS) that is performed by specialized polymerases that synthesize short tracts of DNA opposite lesions, thus enabling reinitiation of replication (2). Error-free bypass mechanisms, mediated by homologous recombination, use an alternative undamaged template to rescue stalled replication forks (3). Stalled replisomes can also be rescued by repriming downstream of the blockage, leaving a gap opposite the lesion (4, 5).Eukaryotes and prokaryotes encode distinct TLS polymerases required for DNA damage tolerance (e.g., Y-family Pols). Although much of our understanding of TLS mechanisms has come from studies of archaeal Y-family DNA polymerases, the majority of archaeal species lack canonical TLS enzymes (Fig. 1A) (6), surprising given the otherwise high degree of conservation between eukaryotic and archaeal replisomes. Many archaea do not appear to encode nucleotide excision repair or photolyase pathways that remove UV light-induced damage (6). These anomalies pose the question as to how archaea, lacking canonical TLS or lesion repair pathways, tolerate the presence of lesions that stall replication. This is particularly pertinent to archaea because of the harsh environmental conditions under which many species reside, including extreme temperatures, which promote increased levels of DNA damage.Open in a separate windowFig. 1.A. fulgidus replisomal enzymes displaying DNA polymerase activity. Analysis of 173 archaeal genomes revealed that only 79 archaea encode canonical TLS DNA polymerases. DNA polymerization activities of A. fulgidus replisomal enzymes. (A) The absence of genes encoding Y-family DNA polymerases in most archaea is shown. (B) Structural elements present in A. fulgidus replisomal enzymes. Abbreviations: AEP, archaeo-eukaryotic primase; CTD, carboxy terminal domain; Exo, exonuclease; NTD, amino terminal domain; Pol, polymerase; and Zn, zinc binding site. (C) Polymerization on nondamaged templates. (D) Single nucleotide incorporation on nondamaged templates. The letter C denotes no enzyme control. The triangles above gel panels indicate time course of the polymerization (30 s, 1'', 5'', and 10'').Archaeal replicases (B- and D-family Pols) specifically arrest at deoxyuracil (dU) (7, 8). This unique feature is limited to replicases from archaea (9). Two important questions regarding dU-induced stalling of archaeal replisomes remain unanswered. First, why do archaea stall replication in response to the template strand dU? Second, how are archaeal genomes containing dU copied? This stalling mechanism may have evolved to prevent promutagenic bypass of the template strand dU, resulting in C–T transition (7, 9). The mechanism used by archaea to resume replication after dU-induced replisome stalling has not been identified.In this study, we report that archaeal replicative primases (primase small subunit, Pri S) can perform translesion DNA synthesis on damaged DNA templates. Pri S can bypass common DNA lesions, such as oxidative and UV damages, faithfully bypassing cyclobutane pyrimidine dimers (CPDs). Additionally, we report that Pri S can replicate past template strand dUs, even in the presence of stalled replicative polymerase B and proliferating cell nuclear antigen (Pol B/PCNA) complexes, thus providing a specific mechanism for maintaining timely replication of DNA containing dU lesions. Together, these findings establish that the archaeal primase is not only required for de novo primer synthesis during initiation of DNA replication but also actively participates during the elongation step by assisting the major DNA replicases in traversing DNA lesions.     

Structure–function analysis of ribonucleotide bypass by B family DNA replicases

Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.Replication of the eukaryotic nuclear genome initiates when RNA primase synthesizes RNA primers of about 10 nucleotides (1). Because this occurs at multiple replication origins and at ∼200-bp intervals on the lagging strand template, about 5% of the genome is initially synthesized as chains of consecutive ribonucleotides. These ribonucleotides are subsequently removed during Okazaki fragment maturation by the combined action of ribonucleases (RNases) H (2) and flap endonucleases (3). Ribonucleotides are also incorporated into DNA by DNA polymerases (Pol) α, δ, and ε, because they discriminate against ribonucleoside triphosphates (rNTPs) efficiently but imperfectly (4) and because cellular rNTP concentrations are much higher than dNTP concentrations (4). As a consequence, large numbers of ribonucleotides are incorporated during replication, and are present in the genomes of cells defective in the repair enzymes that initiate their removal, RNase H2 (5-9) and topoisomerase 1 (10).Ribonucleotides in DNA are a dual-edged sword, in that they have both beneficial and deleterious consequences. On the beneficial side, two consecutive ribonucleotides in the genome are signals for mating type switching in Schizosaccharomyces pombe (11). In addition, recent evidence suggests that RNase H2-dependent processing of ribonucleotides incorporated into the Saccharomyces cerevisiae genome by Pol ε, the primary leading strand replicase, generates a signal that can direct mismatch repair (MMR) to correct replication errors in the nascent leading strand (9, 12). Other possible beneficial signaling roles for ribonucleotides have also been considered (4, 13).On the deleterious edge of the sword, the 2′-oxygen on a ribose sugar in DNA can attack the backbone and render DNA chemically unstable. Yeast strains defective in RNase H2-dependent ribonucleotide excision repair (RER) (5, 14) exhibit several characteristics of replicative stress, including strongly elevated rates for deleting 2–5 bp from repetitive DNA sequences (5, 15), events that are initiated by topoisomerase 1 cleavage of a ribonucleotide in DNA (10, 16). Yeast strains defective in RNase H2 and RNase H1 progress slowly through S phase, accumulate ubiquitylated proliferating cell nuclear antigen (PCNA), and are sensitive to treatment with hydroxyurea (17). Moreover, their survival in the presence of hydroxyurea partly depends on MMS2-dependent template switching and on REV3, which encodes the catalytic subunit of the translesion synthesis (TLS) enzyme Pol ζ. When ribonucleotide incorporation during leading strand replication is increased by a M644G substitution in the Pol ε active site, a defect in RNase H2 results in elevated deletion mutagenesis, elevated dNTP pools, slow growth and activation of the S-phase checkpoint (5, 10, 18), and concomitant deletion of the RNH1 gene encoding RNases H1 is lethal (17). In mice, knocking out any of the genes encoding the three subunits of RNase H2 is embryonic lethal (7, 19). RNase H2 null embryos grow slowly due to reduced cell proliferation and exhibit genome instability and a p53-dependent DNA damage response. Fibroblasts from these embryos contain more than a million single and/or di-ribonucleotides in their genomes and elevated numbers of strand breaks, γ-H2A histone family, member X foci, micronuclei, and chromosomal aberrations. In humans, mutations in the genes encoding RNase H2 are associated with Aicardi-Goutières syndrome, a rare neuroinflammatory condition resembling congenital viral infection (20).These phenotypes of RNase-deficient cells are characteristic of stress that could arise from difficulty in replicating DNA templates containing unrepaired ribonucleotides. This idea is consistent with knowledge that replicases require normal DNA helix geometry to achieve efficient and accurate DNA synthesis, and with crystallographic and NMR studies (21–24) showing that ribonucleotides in DNA alter helix parameters. Recent studies have shown that Pols δ and ε have difficulty bypassing ribonucleotides in DNA templates (4), whereas Pol ζ does not (17). The probability that Pol ε will pause during single ribonucleotide bypass increases after dNTP insertion opposite the ribonucleotide and for several additional insertions opposite deoxynucleotides (5, 25).In this study, we quantify stalling by yeast replicative Pols δ and ε as the number of consecutive ribonucleotides in the DNA template increases from one to four. We show that stalling increases as the number of consecutive ribonucleotides in the DNA template increases, with Pol δ being more efficient at ribonucleotide bypass than Pol ε. We then examine the structural basis for difficulty in ribonucleotide bypass using a homologous B family replicase, bacteriophage RB69 DNA polymerase, as a surrogate that is highly amenable to structural studies (26, 27). To promote the structural analysis, we used a variant of RB69 Pol containing a phenylalanine substituted for Leu415 (28). Leu415 is adjacent to invariant Tyr416, which interacts with the sugar of the incoming dNTP and has an important role in preventing rNTP incorporation (29). An initial crystal structure of L415F RB69 Pol with correctly base-paired dTTP opposite template dA (28) revealed that the phenylalanine ring is accommodated within a cavity present in the WT polymerase without steric clash or major change in active site geometry, consistent with retention of high catalytic efficiency for correct incorporation. Moreover, L415F RB69 Pol can also bypass 8-oxo-guanine more efficiently than can WT RB69 Pol (28). These bypass results were encouraging because the yeast replicases bypass single ribonucleotides with efficiencies somewhat similar to those for bypass of 8-oxo-guanine, and L415F RB69 Pol may therefore facilitate crystallization of ternary complexes with ribonucleotides in the DNA template (25). We show that WT and L415F RB69 Pol also stall during ribonucleotide bypass, to a degree most closely resembling stalling by Pol ε. We describe seven unique crystal structures relevant to L415F RB69 Pol bypass of one or two ribonucleotides. The data are discussed in relation to the consequences of ribonucleotides in the genomes of cells defective in their removal.     

Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.     

Mechanism of replication blocking and bypass of Y-family polymerase {eta} by bulky acetylaminofluorene DNA adducts

Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase η is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol η depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.     

Direct visualization of translesion DNA synthesis polymerase IV at the replisome

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses, we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the β clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the β clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.

Replication fork progression in Escherichia coli is catalyzed by a replisome composed of the DNA polymerase III holoenzyme (Pol III HE), which provides both the leading- and lagging-strand DNA polymerases, the hexameric replicative DNA helicase, DnaB, and the Okazaki fragment primase, DnaG (1). The Pol III HE itself contains 10 subunits: two copies of the core DNA polymerase (αεθ, where α is the catalytic DNA polymerase and ε is the proofreading 3′→5′ exonuclease), the dimeric sliding processivity clamp β, and the DnaX complex, τ₂γδδ′χψ, which loads β to the primer template. Pol III* possess all the subunits except β (1). The core polymerases are bound to the DnaX complex via interaction with the τ subunit (2, 3). An alternative form of the DnaX complex, τ₃δδ′χψ, allows the assembly in vitro of a Pol III HE with three core polymerases (4), the existence of which in vivo is supported by imaging of fluorescently tagged polymerase subunits (5, 6). However, cells that do not produce the γ subunit are ultraviolet (UV) sensitive and have reduced mutagenic break repair (7), an activity that requires DNA polymerase IV (Pol IV) (8).DNA synthesis catalyzed by the Pol III HE is highly accurate, with an error rate of roughly 10⁻⁷ (9). In general, Pol III cannot bypass bulky template lesions, although we have shown that it can bypass a cis-syn thymidine dimer (10). Bypass of most template lesions in the cell is ascribed to the action of translesion DNA synthesis (TLS) polymerases, of which E. coli has three: DNA polymerases II, IV, and V (11). These polymerases demonstrate different activities with various template lesions. Pol V Mut, a RecA-activated form of Pol V (12, 13) [UmuD′₂UmuC (14, 15)], is the major activity under conditions of high replication stress when the SOS response has been activated (16, 17). Pol IV, which is encoded by dinB (18), is a Y family DNA polymerase that is well conserved from bacteria to eukaryotic cells. Pol IV has long been thought to be present at the highest concentration of all DNA polymerases in E. coli under unstressed conditions, about 250 copies per cell, with this concentration increasing about 10-fold upon induction of the SOS response (19). However, a recent study that measured the signal generated by fluorescently tagged Pol IV molecules in live cells argued that these high values were inaccurate, with the basal level of Pol IV being about 20 copies/cell and the SOS-induced level about 280 copies/cell (20).Unlike Pol III HE, which is a rapid and highly processive DNA polymerase (21–23), Pol IV is distributive, incorporating only one nucleotide per primer binding event (24). However, like all E. coli DNA polymerases, it can interact with the β processivity clamp, which increases its processivity to 300–400 nucleotides (24). Overproduction of Pol IV in the absence of replication stress slows DNA replication (25, 26) and it has been demonstrated that it is the main factor contributing to slowing replication fork progression under conditions of stress (27). Studies in vitro have shown that Pol II and Pol IV can generate slow-moving replication forks in the presence of DnaB and DnaG and that very high concentrations of Pol IV can slow the canonical Pol III–DnaB–DnaG replisome (28).The ability of all E. coli TLS polymerases to bind to β led to the formulation of the “tool belt” model to account for rapid localization of TLS polymerases to the site of replisome stalling (29). The conceptual basis of the model was that because β is a dimer, the TLS polymerase could ride along with the replisome on the same sliding clamp that was bound to the α subunit of the Pol III HE and switch places with a stalled Pol III to catalyze lesion bypass. How the polymerase switch occurs is still unclear. It has been suggested that switching occurs only at a stalled polymerase and that the stalled Pol III dissociates from β and is replaced by Pol IV (28, 30, 31). It has also been a common view that Pol IV association with the replisome is concentration dependent (32, 33), accounting for association only when it is needed (i.e., when the SOS response is induced). We have shown that Pol IV–dependent bypass at a thymidine dimer in the leading-strand template in the presence of an active replisome competes with lesion skipping (34), when the stalled leading-strand polymerase cycles ahead to a new primer made on the leading-strand template to continue replication downstream (35), suggesting that polymerase switching had occurred.Here we have addressed association of Pol IV with the replisome by using single-molecule DNA replication (23). We show that in the absence of template damage Pol IV can associate in a concentration-dependent manner with the replisome and proceeds along with it during replication fork progression. Association of Pol IV with the replisome requires its β binding motif, is stabilized by the presence of the γ subunit of the DnaX complex, and generates two classes of replisomes: those without Pol IV bound that progress rapidly and those with Pol IV bound that proceed slowly. Slowing of the replisome by Pol IV does not require its catalytic activity, suggesting that the decrease in rapid replication fork progression is a result of Pol IV binding directly to one of the two polymerase binding clefts on β. The constant presence of Pol IV in the replisome may act as a template damage surveillance mechanism.     

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.Replicative DNA polymerases (DNAPs) from A and B families, collectively termed replicases, exhibit a “tight fit” for their DNA and dNTP substrates and are wondrously adapted to form correct Watson–Crick base pairs, resulting in very pronounced fidelity (1, 2). This strict preference to produce A:T and G:C base pairs is also the Achilles heel of faithful DNA polymerases, however, because they are strongly inhibited by modified nucleotides present at sites of DNA damage, leading to the stalling of replication fork and eventually to replicative stress and cell death (3). At the stalled replication fork, the DNA polymerase may be exchanged by a translesion synthesis (TLS) polymerase, generally belonging to the Y family. These enzymes possess looser solvent-exposed active sites, which allows them to deal with aberrant DNA features much better, although with a very low polymerization accuracy with the risk of the accumulation of mutations and genetic instability (4, 5). Alternatively, nonbulky modified bases, such as uracil and 8-oxo-deoxyguanosine (8oxoG), can be bypassed by replicases, with faithful or mutagenic outcomes that can be modified by the sequence context and dNTP availability (6, 7).Abasic or apurinic/apyrimidinic (AP) sites are the most common DNA lesions arising in cells when the N-glycosydic bond between the sugar moiety and the nucleobase is broken, either spontaneously or by a DNA glycosylase reaction product in the base excision repair pathway (8, 9). Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide selection have been shown to have enhanced AP site bypass capacity (11–15).Among family B DNAPs, protein-primed polymerases constitute a heterogeneous group with an apparent monophyletic origin that can be found in various prokaryotic and eukaryotic viruses, mitochondrial plasmids, and eukaryotic mobile elements (16–19). This designation refers to their capacity, apparent for adenoviruses as well as different bacteriophage enzymes (20, 21), to perform genome replication primed by a specific protein, termed terminal protein (TP), that becomes covalently linked to the 5′ DNA ends. Identification and annotation of these polymerases in databases is based on the presence of two specific amino acid insertions, TPR1 and TPR2, involved in the interaction with TP and in processivity and strand displacement capacity, respectively (22, 23).Bacillus subtilis bacteriophage Φ29 DNA polymerase (Φ29DNAP) is the paradigm of protein-primed DNAPs, with well-understood biochemical and structural properties (reviewed in refs. 18, 20, 24). Φ29DNAP is a highly faithful enzyme (25, 26) able to generate very long DNA molecules (27), coupling DNA synthesis and strand displacement. Φ29, along with other protein-primed genome replication phages, such as PRD1 or Cp-1 (28, 29), can undergo a lytic cycle only after infection of the host cell occurs. In contrast, phage Bam35, which infects Bacillus thuringiensis and related tectiviruses infecting Bacillus cereus sensu lato group (30, 31), are temperate viruses that can self-replicate as linear episomes within lysogenic cells.In this work, we describe the biochemical properties of Bam35 DNA polymerase (B35DNAP) as a faithful, processive DNAP endowed with intrinsic strand displacement activity. Surprisingly, we also found that, despite its high fidelity, it can elude to some extent the tight quality check of proofreading activity, allowing the enzyme to processively bypass abasic sites in DNA. Furthermore, deletion of the TPR2 subdomain does not substantially reduce the insertion of nucleotides opposite the abasic site, but does impair its further extension. We discuss the potential implications of these findings for the bacteriophage replication cycle and possible applications.     

The relationship between dNTP pool levels and mutagenesis in an Escherichia coli NDP kinase mutant

Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia coli results in an increased frequency of spontaneous mutation and an imbalance in dNTP pool levels. It is presumed that the imbalance in dNTP pool levels is responsible for the mutator phenotype of an E. coli ndk mutant. A human homologue of Ndk and potential suppressor of tumor metastasis, nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant. Here, we show that the antimutagenic property of nm23-H2 in E. coli is independent of dNTP pool levels, indicating that dNTP pool imbalance is not responsible for the mutator phenotype associated with the loss of ndk function. We have identified multiple genetic interactions between ndk and genes involved in the metabolism of dUTP, a potentially mutagenic precursor of thymidine biosynthesis. We show that loss of ndk function is synergistic with a dut-1 mutation and synthetically lethal with the loss of thymidine kinase function. Our results suggest that Ndk prevents the accumulation of dUTP in vivo. Based on these results and biochemical studies of Ndk, we propose that the mutagenic phenotype of an ndk mutant is caused by excess misincorporation of uracil in place of thymidine combined with a defect in the uracil base excision pathway.     

A checkpoint control linking meiotic S phase and recombination initiation in fission yeast

During meiosis, high levels of recombination initiated by DNA double-strand breaks (DSBs) occur only after DNA replication. However, how DSB formation is coupled to DNA replication is unknown. We examined several DNA replication proteins for a role in this coupling in Schizosaccharomyces pombe, and we show that ribonucleotide reductase, the rate-limiting enzyme of deoxyribonucleotide synthesis and the target of the DNA synthesis inhibitor hydroxyurea (HU) is indirectly required for DSB formation linked to DNA replication. However, in cells in which the function of the DNA-replication-checkpoint proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, or Cds1p was compromised, DSB formation occurred at similar frequencies in the absence or presence of HU. The DSBs in the HU-treated mutant cells occurred at normal sites and were associated with recombination. In addition, Cdc2p is apparently not involved in this process. We propose that the sequence of meiotic S phase and initiation of recombination is coordinated by DNA-replication-checkpoint proteins.     

Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication

The six main minichromosome maintenance proteins (Mcm2-7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2-7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2-7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Single-molecule analyses revealed that cells with a reduced concentration of Mcm2-7 complexes display normal fork progression but have lost the potential to activate "dormant" origins that serve a backup function during DNA replication. Our data show that the chromatin-bound "excess" Mcm2-7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.     

Regulation of polymerase exchange between Poleta and Poldelta by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

To ensure efficient and timely replication of genomic DNA, organisms in all three kingdoms of life possess specialized translesion DNA synthesis (TLS) polymerases (Pols) that tolerate various types of DNA lesions. It has been proposed that an exchange between the replicative DNA Pol and the TLS Pol at the site of DNA damage enables lesion bypass to occur. However, to date the molecular mechanism underlying this process is not fully understood. In this study, we demonstrated in a reconstituted system that the exchange of Saccharomyces cerevisiae Poldelta with Poleta requires both the stalling of the holoenzyme and the monoubiquitination of proliferating cell nuclear antigen (PCNA). A moving Poldelta holoenzyme is refractory to the incoming Poleta. Furthermore, we showed that the Poleta C-terminal PCNA-interacting protein motif is required for the exchange process. We also demonstrated that the second exchange step to bring back Poldelta is prohibited when Lys-164 of PCNA is monoubiquitinated. Thus the removal of the ubiquitin moiety from PCNA is likely required for the reverse exchange step after the lesion bypass synthesis by Poleta.     

Efficient and accurate bypass of N2-(1-carboxyethyl)-2'-deoxyguanosine by DinB DNA polymerase in vitro and in vivo

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N(2)-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) as the major stable DNA adduct. Here, we found that N(2)-CEdG could be detected at a frequency of one lesion per 10(7) nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N(2)-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N(2)-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N(2)-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase kappa), opposite the N(2)-CEdG is both accurate and efficient. Taken together, our data support that N(2)-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

Activity of the purified mutagenesis proteins UmuC, UmuD'', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.     

Quantitative measurement of translesion replication in human cells: evidence for bypass of abasic sites by a replicative DNA polymerase

Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR is assayed in a subsequent bioassay, by the ability of the plasmid extracted from the cells, to transform an Escherichia coli indicator strain. Using this method it was found that TLR through a synthetic abasic site in the adenocarcinoma H1299, the osteogenic sarcoma Saos-2, the prostate carcinoma PC3, and the hepatoma Hep3B cell lines occurred with efficiencies of 92 +/- 6%, 32 +/- 2%, 72 +/- 4%, and 26 +/- 3%, respectively. DNA sequence analysis showed that 85% of the bypass events in H1299 cells involved insertion of dAMP opposite the synthetic abasic site. Addition of aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilon, caused a 4.4-fold inhibition of bypass. Analysis of two XP-V cell lines, defective in DNA polymerase eta, showed bypass of 89%, indicating that polymerase eta is not essential for bypass of abasic sites. These results suggest that in human cells bypass of abasic sites does not require the bypass-specific DNA polymerase eta, but it does require at least one of the replicative DNA polymerases, alpha, delta, or epsilon. The quantitative TLR assay is expected to be useful in the molecular analysis of lesion bypass in a large variety of cultured mammalian cells.     

Extrachromosomal circular DNA is common in yeast

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.Copy number variants (CNVs) are alterations in number of copies of particular genes or other extensive DNA sequences in a genome. Gene deletions and amplifications are important sources of genetic variation that have proven important in the evolution of multicellular organisms. The high prevalence of families of paralogous genes, such as globins, protein kinases, and serine proteases, strongly supports the idea that gene duplication and divergence underlie much of evolution (1). In human somatic cells, CNVs have been implicated in cancer (2) and aging (3), as well as developmental and neurological diseases (4, 5). The best-studied types of CNVs are chromosomal gene amplifications and deletions that can easily be characterized by genome sequencing or tiling arrays (6, 7) or as cytological visible homogeneously staining regions (8).Extrachromosomal circular DNAs (eccDNAs) represent a less studied form of CNVs although examples of eccDNAs have been detected in many organisms. In humans, eccDNAs are produced during Ig heavy-chain class switching (9). In higher plant genomes, repeat-derived eccDNAs are considered intermediates in the evolution of satellite repeats (10). In aging Saccharomyces cerevisiae cells, ribosomal rDNA circles accumulate (11). Also, in yeast, eccDNAs are generated from telomeric regions that contain many repeated sequences (12), and recombination between retrotransposon or their remnants in the form of solo long terminal repeats (LTRs) has been implicated in the formation of eccDNA (13, 14). Generally, eccDNAs are acentric and thus expected to missegregate during cell division, resulting in copy number variation (15). However, the abundance of eccDNAs and their role in evolution remains unknown.Simple recombinational models have been proposed that connect duplications, deletions, and eccDNA with one another. In the simplest model, a gene deletion can be produced by intrachromatid ectopic recombination between tandem repeats that flank the same gene, simultaneously producing a circular DNA element (Fig. 1A) (14). CNVs are also produced by other mechanisms that do not require a circular intermediate, such as replication slippage (16), interchromatid or interchromosome recombination between nonallelic homologs (17). However, DNA studies of several loci in human germ cells suggest that the frequency of intrachromatid ectopic recombination, which leads to eccDNA, could be just as high as other CNV events (18). Incorrect nonhomologous end joining of DNA ends can generate eccDNA (Fig. 1A) (2, 19),) in addition to other mechanisms (Fig. 1A). These mechanisms could include microhomology-mediated DNA repair processes at double-strand DNA breaks (20, 21), as well as processes independent of DNA breaks, such as replication errors near short inverted repeats (22) or small single-stranded DNA that prime formation of eccDNA (23).Open in a separate windowFig. 1.Genome-wide eccDNA measurement by Circle-Seq. (A) Models of extrachromosomal circular DNA (eccDNA) formation: intrachromatid ectopic homologous recombination (HR), nonhomologous end-joining (NHEJ), and other circularization mechanisms. (B) Circle-Seq procedure. From disrupted S. cerevisiae cells, eccDNA was purified by (1) column purification for circular DNA, (2) digestion of remaining linear DNA by plasmid-safe ATP-dependent DNase facilitated by NotI endonuclease, and (3) rolling-circle amplification by ϕ29 DNA polymerase and subsequent high-throughput DNA sequencing. (C) Sequenced 141-nucleotide reads were mapped to the S288C S. cerevisiae reference genome. Shown are data from reference sample R3 for part of chromosome IV. Green, paralogous genes; black, unique genes or ORFs; blue, read coverage mapped to the chromosome; gray boxes, individual reads with homology to the Watson (+) (light gray) and Crick (−) (dark gray) strands. (D) EccDNA was identified from regions covered by contiguous mapped reads >1 kb that were found to be significantly overrepresented by Monte Carlo simulation. Black bars, chromosomal regions recorded as eccDNAs; white bars, chromosomal regions excluded from the analysis (contiguous reads <1 kb).There is likely an intimate connection between deletions, duplications, and circular DNA forms: reintegration of a circular copy of a gene by homologous recombination results in a duplication, just as recombination between duplicates can generate circular DNA copies and deletions. Chromosomal CNVs have been generally detected well after their establishment. Thus, the mechanism for formation of copy number variable regions can be inferred only from their structure (24, 25). Detection of new, potentially transient chromosomal duplications is challenging because it involves detection of alterations in single DNA molecules within large cell populations. We reasoned that detection and recovery of eccDNAs might be more tractable than other CNVs because of the unique and well-studied biophysical and biochemical properties of circular DNA molecules.We developed a sensitive, genome-scale enrichment and detection method for eccDNA (Circle-Seq), based on well-established prokaryotic plasmid purification (26, 27) and deep sequencing technology. With Circle-Seq, we were able to purify, sequence, and map eccDNAs that, among them, cover nearly a quarter of the whole genome sequence of S. cerevisiae. We infer from the numerous eccDNA findings that circularization of genomic sequences is common enough to account for the generation of much of the variation in gene copy numbers observed in cancer and other human genetic diseases, as well as the process of evolution by duplication, deletion, and divergence.     

A model for DNA polymerase switching involving a single cleft and the rim of the sliding clamp

The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the β sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883–5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303–P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.     

rDNA array length is a major determinant of replicative lifespan in budding yeast

The complex processes and interactions that regulate aging and determine lifespan are not fully defined for any organism. Here, taking advantage of recent technological advances in studying aging in budding yeast, we discovered a previously unappreciated relationship between the number of copies of the ribosomal RNA gene present in its chromosomal array and replicative lifespan (RLS). Specifically, the chromosomal ribosomal DNA (rDNA) copy number (rDNA CN) positively correlated with RLS and this interaction explained over 70% of variability in RLS among a series of wild-type strains. In strains with low rDNA CN, SIR2 expression was attenuated and extrachromosomal rDNA circle (ERC) accumulation was increased, leading to shorter lifespan. Suppressing ERC formation by deletion of FOB1 eliminated the relationship between rDNA CN and RLS. These data suggest that previously identified rDNA CN regulatory mechanisms limit lifespan. Importantly, the RLSs of reported lifespan-enhancing mutations were significantly impacted by rDNA CN, suggesting that changes in rDNA CN might explain the magnitude of some of those reported effects. We propose that because rDNA CN is modulated by environmental, genetic, and stochastic factors, considering rDNA CN is a prerequisite for accurate interpretation of lifespan data.

Budding yeast, Saccharomyces cerevisiae, has been a foundational model organism for the study of cellular aging. Cells divide asymmetrically and the mother cell undergoes a limited number of divisions, which defines the cell’s replicative lifespan (RLS). Measuring RLS is technically challenging and only recent advances have enabled more efficient screening for lifespan-modulating factors (1–5).A number of processes, pathways, and mechanisms have been implicated in yeast aging (reviewed in ref. 6), with regulators of the ribosomal RNA gene array (rDNA) representing perhaps the best-characterized group of lifespan modulators. Proteins that act at the rDNA locus modify rDNA stability and the formation of extrachromosomal rDNA circles (ERCs), a known aging factor in S. cerevisiae (7). Sir2, the defining member of the Sirtuin family of histone deacetylases, silences the rDNA locus and suppresses formation of ERCs (8). Conversely, the protein Fob1 binds to a replication fork barrier site in the rDNA locus, decreases rDNA stability, and thus promotes the production of ERCs (9, 10). Since the accumulation of ERCs in the mother cell limits its RLS, sir2Δ and fob1Δ strains are short and long lived, respectively (8, 11).The rDNA locus is highly repetitive and dynamic. While 150 repeats are considered a normal copy number (CN) for the strain background used in this study, the number of repeats commonly ranges from 100 to 250 copies (12). And while the size of the rDNA array is relatively stable for a limited number of divisions, rDNA CN can change on a timescale faster than the estimated mutation rate (13). Thus, it may be considered a type of “contingency locus,” which is characterized by environmentally responsive genetic variation that results in distinct phenotypic outcomes (14–16).The rDNA copy number varies significantly in strains found in the wild and those used in the laboratory (12, 13, 17–19). This variation can occur spontaneously, but can also be introduced by standard laboratory DNA transformation protocols or changes in growth environment (12, 13, 20). In addition, there are Sir2- and Fob1-dependent feedback mechanisms in place by which a “normal” rDNA CN is maintained through modulation of Sir2 expression levels (21). Interestingly, rDNA array size is anticorrelated with ERC abundance in young cells, suggesting that chromosomal and extrachromosomal rDNA are in an equilibrium (13). Despite this known connection between rDNA array size and Sir2 and ERC levels, no evidence for array size impacting lifespan has been found (12, 15, 18).Yet, significant variability exists within reported lifespans of S. cerevisiae. A metastudy found that the RLS of the same wild-type strains varied between 20 and 40, depending on the study in which it was reported (22). This variability was attributed to a reporting bias and small sample sizes. However, these findings are also consistent with uncontrolled genetic or environmental factors contributing to the variability. In addition, a genome-wide screen measuring the RLS of deleted nonessential genes found a significant discrepancy between strains with opposing mating types carrying the same gene deletion (23). This discrepancy was mostly attributed to statistical error due to a low number of cells analyzed, but was not fully explored. Given this large degree of variability, which likely impacts the interpretation of any lifespan measurement, we wanted to address the underlying cause. Here we show that the chromosomal rDNA copy number is an important determinant of replicative lifespan in yeast and can explain a large part of the reported variability in lifespan.     

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. In this report we show that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila melanogaster CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and we suggest that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage.