RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.     

Solution NMR Structure of the DNA-binding Domain from Scml2 (Sex Comb on Midleg-like 2)

Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multisubunit protein complex, PRC1 (Polycomb repressive complex 1), which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the “stemness” of the cell, and cell differentiation. Although the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. In this work a conserved protein domain within human Scml2 has been identified, and its structure was determined by solution NMR spectroscopy. This module was named Scm-like embedded domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by malignant brain tumor domain (MBT) and sterile α motif (SAM) domains. The domain adopts a novel α/β fold with no structural analogues found in the Protein Data Bank (PDB). The ability of the SLED to bind double-stranded DNA was also examined, and the isolated domain was shown to interact with DNA in a sequence-specific manner. Because PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes.     

Analysis of the Activities of RAD54, a SWI2/SNF2 Protein,Using a Specific Small-molecule Inhibitor

RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.     

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7–11.2-kbp linear duplex DNAs at a rate of ∼250 bp s⁻¹, while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp⁻¹. Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the “leading” AdnB motor propagates a Y-fork by translocation along the 3′ DNA strand, ahead of the “lagging” AdnA motor domain. By tracking the resection of the 5′ and 3′ strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5′ strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3′ strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.     

Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.     

Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins

Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3'→5' direction, whereas RuvAB drives it in the 5'→3' direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.     

The Role of the Human Psoralen 4 (hPso4) Protein Complex in Replication Stress and Homologous Recombination

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G₂/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

Structure and cellular dynamics of Deinococcus radiodurans single-stranded DNA (ssDNA)-binding protein (SSB)-DNA complexes

The single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair.     

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.     

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg²⁺-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.     

Crystal Structure of the Ubiquitin-like Domain-CUT Repeat-like Tandem of Special AT-rich Sequence Binding Protein 1 (SATB1) Reveals a Coordinating DNA-binding Mechanism

SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1–248, SATB1^(1–248)), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.     

Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM

MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe¹¹⁴ and Met⁷⁷ in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met⁷⁷ plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe¹¹⁴, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair.     

Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment

To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors.     

Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA and Mediates Annealing of Complementary Strands Coated by SsbB and SsbA

Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.     

The RING Finger Protein RNF8 Ubiquitinates Nbs1 to Promote DNA Double-strand Break Repair by Homologous Recombination

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.     

Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a recombination mediator to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.