Ataxia telangiectasia-mutated protein and DNA-dependent protein kinase have complementary V(D)J recombination functions

Antigen receptor variable region exons are assembled during lymphocyte development from variable (V), diversity (D), and joining (J) gene segments. Each germ-line gene segment is flanked by recombination signal sequences (RSs). Recombination-activating gene endonuclease initiates V(D)J recombination by cleaving a pair of gene segments at their junction with flanking RSs to generate covalently sealed (hairpinned) coding ends (CEs) and blunt 5'-phosphorylated RS ends (SEs). Subsequently, nonhomologous end joining (NHEJ) opens, processes, and fuses CEs to form coding joins (CJs) and precisely joins SEs to form signal joins (SJs). DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activates Artemis endonuclease to open and process hairpinned CEs before their fusion into CJs by other NHEJ factors. Although DNA-PKcs is absolutely required for CJs, SJs are formed to variable degrees and with variable fidelity in different DNA-PKcs-deficient cell types. Thus, other factors may compensate for DNA-PKcs function in SJ formation. DNA-PKcs and the ataxia telangiectasia-mutated (ATM) kinase are members of the same family, and they share common substrates in the DNA damage response. Although ATM deficiency compromises chromosomal V(D)J CJ formation, it has no reported role in SJ formation in normal cells. Here, we report that DNA-PKcs and ATM have redundant functions in SJ formation. Thus, combined DNA-PKcs and ATM deficiency during V(D)J recombination leads to accumulation of unjoined SEs and lack of SJ fidelity. Moreover, treatment of DNA-PKcs- or ATM-deficient cells, respectively, with specific kinase inhibitors for ATM or DNA-PKcs recapitulates SJ defects, indicating that the overlapping V(D)J recombination functions of ATM and DNA-PKcs are mediated through their kinase activities.     

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.     

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5′→3′ double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.     

From the Cover: PNAS Plus: Single-molecule visualization of RecQ helicase reveals DNA melting,nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.DNA helicases are ubiquitous enzymes involved in many aspects of DNA metabolism, including DNA replication, repair, and recombination. These enzymes work by coupling the hydrolysis of nucleoside triphosphates (NTPs) to unwinding of double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) (1). This activity allows the cell’s machinery to access the information stored within the bases of the double helix. RecQ helicase from Escherichia coli is the founding member of the RecQ family of helicases (2). These enzymes belong to the superfamily 2 (SF2) group of helicases, yet share greater sequence homology with their own family members, and they play important roles in the maintenance of genomic integrity by DNA recombination and repair (1, 3). Mutations in the human RecQ-like helicases, Bloom (BLM), Werner (WRN), and RecQ4 proteins, lead to Bloom’s, Werner’s, and Rothmund–Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancers (4).E. coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2, 5, 6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7–10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7, 9, 11, 12). RecQ also functions with topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate DNA molecules and to separate converged replication forks (13, 14). RecQ and Topo III provide an alternative to the RuvABC pathway for disengaging double Holliday junctions and do so without producing chromosomal crossovers (15).In vitro, RecQ can unwind a multitude of DNA substrates and does not require a ssDNA tail, or even a dsDNA end, to initiate unwinding; consequently, it is distinctive in being able to unwind covalently closed circular plasmid DNA (16, 17). The winged-helix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures. A curious feature of RecQ is that maximal unwinding requires nearly stoichiometric amounts of protein relative to the DNA (one protein per ∼10 bp), which can be partially mitigated (one protein per ∼30 bp) by including the ssDNA binding protein, SSB (5, 6, 16, 19). This behavior is compatible with the low processivity of ssDNA translocation (∼30–100 nucleotides) by RecQ (20–22) and other RecQ members (23). Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9, 16, 24–26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27–29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30–35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments. In particular, total internal reflection fluorescence (TIRF) microscopy permits the detection of individual fluorophores with high sensitivity in the presence of a high background (30). To visualize the activity of DNA binding proteins and helicases, TIRF microscopy can be coupled with microfluidic techniques to facilitate visualization of molecules and exchange of solution components (36–39).Ensemble assays have elucidated many features of DNA unwinding by the RecQ helicase family, but the need to average over a heterogeneous and unsynchronized population of enzymes has precluded a thorough understanding of this diverse and universally important helicase family. To permit a more insightful analysis, we directly visualized unwinding of individual molecules of DNA by RecQ helicase. Unwinding was monitored using fluorescent SSB to visualize generation of ssDNA. On single DNA molecules, we could see tracks of the fluorescent SSB binding to ssDNA produced by the helicase activity of RecQ. We show that RecQ initiates DNA unwinding via melting of duplex DNA at internal sites. Once initiated, DNA unwinding propagates either uni- or bidirectionally via the cooperative action of multiple RecQ molecules at the junction of ssDNA with dsDNA. Collectively, these observations define a stable oligomeric complex of subunits involved in processive helicase action, which is concordant with both biochemical and biological function of RecQ helicases and other helicases.     

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4^−/− cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.DNA double-strand breaks (DSBs) are one of the most severe forms of DNA damage that can result from pathological conditions such as replication stress, exposure to ionizing radiation (IR), free radicals, or other DNA-damaging drugs or because of failed single-strand break repair (SSBR) (1, 2). In developing lymphocytes, programmed DSBs are essential intermediates for antigen receptor gene rearrangements, including V(D)J recombination and Ig heavy chain class switch recombination (CSR) (1, 2). Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two major pathways for DSB repair. Whereas HR is restricted to the S/G₂ phase of the cell cycle, NHEJ is active throughout the cell cycle and is generally considered the major pathway for DSB repair in mammals (1, 2).The NHEJ pathway has been extensively studied. The core components include the Ku70/Ku86 heterodimer, DNA-dependent protein kinase, X-ray cross complementation factor 4 (XRCC4), and DNA ligase IV (Lig4) (1, 2). Additional NHEJ factors include the Artemis nuclease, XRCC4-like factor (XLF) (or Cernunnos), Paralog of XRCC4 and XLF, and Polymerases µ and λ. Missing any of these factors results in various degrees of DSB repair deficits that are highly context-dependent. In general, cells lacking core components of NHEJ are hypersensitive to IR and abolished for V(D)J recombination but are only partially defective for CSR and competent for circulation of transfected linearized plasmids, suggesting that there exists an “alternative” way to join at least some types of DSBs. This alternative end-joining (A-EJ) pathway has recently become a focal area of research because of its implications in oncogenic chromosomal translocations (3), which are rare in NHEJ-proficient cells but much more frequent when NHEJ is compromised. Little is known about A-EJ other than it is kinetically slow and uses an increased level of microhomology (nucleotide overlaps that can be assigned to either of the two DNA ends) during joining (2, 4). A number of DNA repair factors, many of which are involved in SSBR, have been implicated in A-EJ (5), but the overall composition of A-EJ remains elusive. It is still unclear whether A-EJ is a distinct pathway, consists of multiple subpathways, or is merely an aberrant form of NHEJ with missing components substituted by compatible but less efficient factors. It is also unclear whether A-EJ contributes to DSB repair in NHEJ-proficient cells at all or is only active when NHEJ is compromised.Much of our understanding of mechanistic details of DSB repair has derived from studies of V(D)J recombination and CSR; both involving DSB intermediates (1). V(D)J recombination is initiated by the recombination-activating genes (RAGs) that bind and cleave at specific DNA sequences flanking the V, D, and J segments to assemble an exon encoding the variable (antigen binding) domain of the B- and T-cell receptors. CSR is initiated by activation-induced cytidine deaminase (AID) in antigen-stimulated B cells that changes the IgH constant (C) region to a different isotype. AID catalyzes DNA cytosine deamination (converting cytosines to uracils) at switch regions preceding each C region (6, 7). Processing of AID-generated uracils, through a mechanism still not fully characterized, leads to DSB formation. Although both processes use NHEJ to join DSBs, in cells missing any of the core components of NHEJ, CSR is only partially defective, whereas V(D)J recombination is completely abolished. It has been reported that the RAG complex holds the four broken ends in a postcleavage complex and directs VDJ-associated DSBs into the NHEJ pathway (8, 9). In contrast, significant levels of CSR can occur in the absence of any core NHEJ factors (10, 11–14), suggesting that switch region breaks are more accessible to alternative DSB repair pathways.Regardless of how broken DNA ends are processed, at least one DNA ligase is required to ligate the two ends. Vertebrates have three ATP-dependent DNA ligases (Lig1, Lig3, and Lig4) (15). Lig1 and Lig4 are conserved in all eukaryotes, whereas Lig3 is only present in vertebrates (15). Lig4 is a core component of the NHEJ pathway and functions exclusively in NHEJ. Cells deficient for Lig4, or its cofactor XRCC4, display the most severe phenotypes of NHEJ deficiency. In the absence of Lig4, A-EJ must rely on Lig1 or Lig3 (or both). It is generally accepted that the major role of Lig1 is to join Okazaki fragments during DNA replication, and this function is mediated by an interaction with the proliferating cell nuclear antigen (PCNA) (16). Lig3 is produced in somatic cells in two forms (mitochondrial and nuclear) via alternative translation initiation (17). It was recently shown that mitochondrial, but not nuclear, Lig3 is essential for cell viability (18, 19). Nuclear Lig3 stably interacts with the X-ray complementation factor 1 (XRCC1), a scaffold protein that is essential for base excision repair. For this reason, Lig3 is regarded as the primary DNA repair ligase for SSBR, although Lig1 has also been implicated in DNA repair (15).We have previously reported the disruption Lig4 and Lig1 in a mouse B-cell line (CH12F3) capable of robust cytokine-induced CSR (10). Lig4^−/− CH12F3 cells undergo kinetically slow but significant levels of CSR [∼50% of wild-type (WT) level at day 3] (10). Switch junctions isolated from Lig4^−/− CH12F3 cells show increased microhomology and no direct joins (10). In the present study, we focused on determining which of two remaining ligases (Lig1 and Lig3) is responsible for A-EJ during CSR in Lig4^−/− cells. To that end, we disrupted Lig1 and Lig3 (nuclear) individually in Lig4^−/− CH12F3 cells. We found that Lig1 and Lig3 have redundant functions in DNA repair in response to a variety of DNA-damaging agents and during repair by A-EJ during CSR.     

Completion of DNA replication in Escherichia coli

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.During chromosomal replication, cells tightly regulate the processes of initiation, elongation, and completion to ensure that each daughter cell inherits an identical copy of the genetic information. Although the mechanisms regulating initiation and elongation have been well characterized (reviewed in refs. 1, 2), the process of how cells recognize replicated regions and complete replication at the precise doubling point remains a fundamental question yet to be addressed. Whether this event occurs once per generation as in Escherichia coli or thousands of times per generation as in human cells, the failure to efficiently carry out this function would be expected to result in a loss of genomic stability. Considering the large number of proteins that cells devote to ensuring the fidelity of replication initiation and elongation, it seems highly probable that the final critical step in this process will be also be tightly regulated and controlled enzymatically.In some aspects, one could argue that the efficiency of completion is likely to be more critical to the faithful duplication of the genome than that of initiation. When replication origins fail to initiate efficiently, elongation of replication forks from neighboring origins is often able to compensate (3, 4), and both prokaryotic and eukaryotic cells are able to tolerate variations in their origin number without severe phenotypic consequences (5–7). However, a failure to accurately limit or join any event where forks converge would be expected to result in duplications, deletions, rearrangements, or a loss of viability depending upon how the DNA ends are resolved at segregation.A number of studies suggest that an ability to sense when all sequences in the genome have doubled is critical to genomic replication. In vitro, converging replisomes continue through their meeting point as one replisome displaces the other, resulting in over-replication, or a third copy, of the region where the forks meet (8). Complicating the process of genomic doubling even further, several studies have suggested that illegitimate initiations of replication frequently occur at single-strand nicks, gaps, D-loops, and R-loops throughout the genomes of both prokaryotes and eukaryotes (9–14). Similar to when replication forks continue through a previously replicated template, each of these events would generate a third copy of the chromosomal region where the event occurs. Thus, over-replication may be inherent and promiscuous during the duplication of genomes. If true, then to ensure that each sequence of the genome replicates once, and only once, per generation, cells must encode an enzymatic system that is essentially able to count in pairs and efficiently degrade odd or over-replicated regions until the two nascent end pairs of replication events can be joined.The model organism E. coli is particularly well-suited to dissect how this fundamental process occurs. In E. coli, the completion of replication occurs at a defined region on the genome, opposite to the bidirectional origin of replication (15). Most completion events can be further localized to one of six termination (ter) sequences within the 400-kb terminus region due to the action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner, in effect stalling the replication fork at this site until the second arrives (16, 17). Although Tus confines converging replication forks to a specific region, it does not appear to be directly involved in the completion reaction because tus mutants have no phenotype and complete replication normally (18). Furthermore, plasmids and bacteriophage lacking ter sequences are maintained stably (19).Many mutants impaired for either replication initiation or elongation were initially isolated based on their growth defects or an impaired ability to maintain plasmids (20–22). We reasoned that mutants impaired for the ability to complete replication might be expected to exhibit similar phenotypes and initially focused our attention on the properties of recBC and recD mutants. RecB-C-D forms a helicase–nuclease complex that is required for homologous repair of double-strand breaks in E. coli (23, 24). The enzyme uses specific DNA sequences, termed “Chi sites,” to initiate recombination between pairs of molecules. Loss of RecB or C inactivates the enzyme complex, whereas loss of RecD inactivates the nuclease and Chi recognition, but retains helicase activity (23, 24). Here, we show that inactivation of RecBCD leads to a failure to recognize and join replicating molecules at their doubling point. Although the completion process requires RecBCD, it is distinct from double-strand break repair and does not involve a double-strand break intermediate, homologous recombination, or RecA.     

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.Helicases are motor proteins that convert the chemical energy of nucleoside triphosphate (NTP) hydrolysis into the mechanical energy needed to separate nucleic acid strands (1). The largest and most diverse helicase superfamilies, SF1 and SF2, use conserved sequence motifs (I, Ia, II–VI) within their helicase domains to couple NTP hydrolysis to conformational changes that mediate DNA translocation and unwinding (1, 2). Although the DNA-unwinding mechanisms of SF1 helicases have been examined extensively, far less is known about SF2 enzymes, in part because of the smaller number of available helicase/substrate complex structures.RecQ DNA helicases are SF2 enzymes with broad roles in promoting genomic stability in eubacterial and eukaryotic species (3). Their importance is underscored by the multiple genomic instability diseases caused by mutations in human recQ genes (4–8). At a structural level, most RecQ helicases share a similar domain architecture that includes a helicase domain, a RecQ C-terminal (RQC) element comprised of Zn²⁺-binding and winged-helix (WH) domains, and a helicase and RNaseD C-terminal (HDRC) domain (Fig. 1A) (9, 10). The helicase and RQC domains combine to form a catalytic core that is sufficient for DNA-unwinding activity in many RecQ proteins. Crystal structures of several RecQ catalytic cores have been determined, including Escherichia coli RecQ (EcRecQ), human RecQ1, and human Bloom syndrome protein (BLM) [refs. 10–12 and unpublished structures (4CDG, 2WWY, and 4CGZ) available through the Protein Data Bank (PDB)]. A comparison of these structures reveals strong similarities among domains within the catalytic core but also differences in the relative positioning of these domains among RecQ proteins. The EcRecQ and antibody-bound BLM structures form an open arrangement in which the WH domain is centered relative to the helicase and Zn²⁺-binding domains, but in RecQ1 and DNA-bound BLM a closed arrangement is observed with the WH domain positioned laterally to the helicase domain (Fig. 1B and Fig. S1) (10–12). The functional relevance of the open and closed arrangements is unknown.Open in a separate windowFig. 1.Structure of the DNA-bound CsRecQ catalytic core. (A) Schematic diagram of RecQ DNA helicase. Coloring denotes the helicase (blue and red), Zn²⁺-binding (yellow), WH (green), and HRDC (gray) domains. (B) Overlay of RecQ structures highlights the mobility of the WH domain. (C) Ribbon diagram of the CsRecQ/DNA crystal structure. Domains are colored as in A with DNA colored orange. A labeled schematic of the DNA is shown with disordered nucleotides as dots.Although several RecQ structures have been defined, insights into the mechanisms by which this important helicase family recognizes and unwinds DNA substrates have been hampered by limitations in reported RecQ/DNA complex structures. One outstanding question is how RecQ enzymes can unwind diverse DNA substrates. Most RecQ enzymes, including EcRecQ, unwind a very broad array of substrates that include duplex, triplex, quadruplex, and branched DNA (3). Human RecQ1, which unwinds Holliday junction but not triplex or quadruplex DNA, is a rare exception in its narrow substrate specificity (13). The mechanisms that confer functional diversity in most, but not all, RecQ enzymes remain unknown. A second question is how RecQ enzymes couple DNA binding with DNA-dependent ATPase functions. A chemo-mechanical coupling element called the “aromatic-rich loop” (ARL) is encoded C-terminal to motif II in RecQ proteins (10, 14), but how DNA binding at the ARL is linked to ATPase functions is unclear because current RecQ/DNA structures have not resolved the DNA/ARL interface. ARL-coupling elements have been examined in several SF1 helicases, but they map to different positions within the helicase domain and rely on structural features that are not found in RecQ enzymes (15–21), leaving their mechanistic similarity with ARLs in RecQ enzymes unclear. A third puzzling feature of RecQs is their varied use of a WH domain β-hairpin in DNA unwinding. In eukaryotic RecQ helicases, this β-hairpin projects away from the WH domain, forming a wedge that appears to separate DNA strands (11, 12, 22). However, the equivalent β-hairpin in EcRecQ is much shorter and is dispensable for helicase activity (10, 11), indicating that the bacterial and human RecQ proteins use different strand-separation mechanisms. This finding is even more surprising given that putative wedge elements have been identified in all other SF1 and SF2 DNA helicases of known structure (23). Understanding how bacterial RecQs function without a wedge element could offer new insights into the mechanisms by which helicases can unwind DNA.In this report, the structure of a bacterial RecQ catalytic core bound to duplex DNA with a 3′ single-stranded extension provides insights into physical mechanisms of RecQ enzymes. DNA binding induces a rearrangement of the RecQ catalytic core in which the WH pivots ∼90° from an open to a closed conformation to secure duplex DNA against a surface of the helicase domain. Flexibility in the WH domain position could provide the structural plasticity needed for RecQ to accommodate diverse substrates in its DNA-binding site. A second conformational change remodels the ARL to allow ssDNA binding. This DNA-dependent conformational shift triggers reorganization of the ATP-binding pocket in a manner that positions active-site residues for catalysis, explaining how the ARL couples DNA binding and ATPase activities in RecQ enzymes. The DNA within the complex is bent at a 90° angle at the unwinding junction, and this sharp bend could explain the ability of bacterial RecQs to unwind DNA without the use of a wedge.     

Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity

The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5′-ended strand to form 5′ mononucleotides and a long 3′ overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5′-ended strand insert into the active site of one subunit of the trimer, while the 3′-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5′ phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg²⁺ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5′ phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.     

Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells

Genomic integrity often is compromised in tumor cells, as illustrated by genetic alterations leading to loss of heterozygosity (LOH). One mechanism of LOH is mitotic crossover recombination between homologous chromosomes, potentially initiated by a double-strand break (DSB). To examine LOH associated with DSB-induced interhomolog recombination, we analyzed recombination events using a reporter in mouse embryonic stem cells derived from F1 hybrid embryos. In this study, we were able to identify LOH events although they occur only rarely in wild-type cells (≤2.5%). The low frequency of LOH during interhomolog recombination suggests that crossing over is rare in wild-type cells. Candidate factors that may suppress crossovers include the RecQ helicase deficient in Bloom syndrome cells (BLM), which is part of a complex that dissolves recombination intermediates. We analyzed interhomolog recombination in BLM-deficient cells and found that, although interhomolog recombination is slightly decreased in the absence of BLM, LOH is increased by fivefold or more, implying significantly increased interhomolog crossing over. These events frequently are associated with a second homologous recombination event, which may be related to the mitotic bivalent structure and/or the cell-cycle stage at which the initiating DSB occurs.     

E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks

Hereditary cases of breast and ovarian cancer are often attributed to germ-line mutations of the BRCA1 tumor suppressor gene. Although BRCA1 is involved in diverse cellular processes, its role in the maintenance of genomic integrity may be a key component of its tumor suppression activity. The protein encoded by BRCA1 interacts in vivo with the related BARD1 protein to form a heterodimeric complex that acts as a ubiquitin E3 ligase. Because the enzymatic activity of the BRCA1/BARD1 heterodimer is conserved over a broad phylogenetic range, it is thought to be critical for the central functions of BRCA1. To test this hypothesis, we have generated isogenic clones of embryonic stem cells that do or do not express an enzymatically proficient Brca1 polypeptide. Surprisingly, cells lacking the ubiquitin ligase activity of BRCA1 are viable and do not accumulate spontaneous cytogenetic rearrangements. Gene targeting efficiencies are modestly reduced in these cells, and chromosomal rearrangements arise at elevated rates in response to genotoxic stress. Nonetheless, cells lacking Brca1 enzymatic activity are not hypersensitive to the DNA cross-linking agent mitomycin C. They also form Rad51 focus in response to ionizing radiation and repair chromosome breaks by homologous recombination at wild-type levels. These results indicate that key aspects of BRCA1 function in genome maintenance, including its role in homology-directed repair of double-strand DNA breaks, do not depend on the E3 ligase activity of BRCA1.     

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data.     

Real-time analysis of double-strand DNA break repair by homologous recombination

The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.     

RecQ helicases and topoisomerase III in cancer and aging

RecQ helicases have in recent years attracted increasing attention due to the important roles they play in maintaining genomic integrity, which is essential for the life of a cell and the survival of a species. Humans with mutations in RecQ homologues are cancer prone and suffer from premature aging. A great effort has therefore been made to understand the molecular mechanisms and the biological pathways, in which RecQ helicases are involved. It has become clear that these enzymes work in close concert with DNA topoisomerase III, and studies in both yeast and mammalian systems point to a role of the proteins in processes involving homologous recombination. In this review we discuss the genetic and biochemical evidence for possible functions of RecQ helicases and DNA topoisomerase III in multiple cellular processes such as DNA recombination, DNA replication, and cell cycle checkpoint control. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional redundancy between repair factor XLF and damage response mediator 53BP1 in V(D)J recombination and DNA repair

The classical nonhomologous DNA end-joining (C-NHEJ) double-strand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histone H2AX and 53BP1, which are assembled into chromatin complexes around DSBs. In mice, C-NHEJ, V(D)J recombination, and lymphocyte development are, at most, modestly impaired in the absence of XLF or ATM, but are severely impaired in the absence of both. Redundant functions of XLF and ATM depend on ATM kinase activity; correspondingly, combined XLF and H2AX deficiency severely impairs V(D)J recombination, even though H2AX deficiency alone has little impact on this process. These and other findings suggest that XLF may provide functions that overlap more broadly with assembled DSB response factors on chromatin. As one test of this notion, we generated mice and cells with a combined deficiency for XLF and 53BP1. In this context, 53BP1 deficiency, although leading to genome instability, has only modest effects on V(D)J recombination or lymphocyte development. Strikingly, we find that combined XLF/53BP1 deficiency in mice severely impairs C-NHEJ, V(D)J recombination, and lymphocyte development while also leading to general genomic instability and growth defects. We conclude that XLF is functionally redundant with multiple members of the ATM-dependent DNA damage response in facilitating C-NHEJ and discuss implications of our findings for potential functions of these factors.