Proteomic dissection of DNA polymerization

DNA polymerases replicate the genome by associating with a range of other proteins that enable rapid, high-fidelity copying of DNA. This complex of proteins and nucleic acids is termed the replisome. Proteins of the replisome must interact with other networks of proteins, such as those involved in DNA repair. Many of the proteins involved in DNA polymerization and the accessory proteins are known, but the array of proteins they interact with, and the spatial and temporal arrangement of these interactions, are current research topics. Mass spectrometry is a technique that can be used to identify the sites of these interactions and to determine the precise stoichiometries of binding partners in a functional complex. A complete understanding of the macromolecular interactions involved in DNA replication and repair may lead to discovery of new targets for antibiotics against bacteria and biomarkers for diagnosis of diseases, such as cancer, in humans.     

Single-molecule studies of DNA replisome function

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.     

DNA damage detection by an archaeal single-stranded DNA-binding protein

Archaeal DNA repair pathways are not well defined; in particular, there are no convincing candidate proteins for detection of DNA mismatches or the bulky lesions removed by excision repair pathways. Single-stranded DNA-binding proteins (SSBs) play a central role in DNA replication, recombination and repair. The crenarchaeal SSB is a monomer with a single oligonucleotide-binding fold for single-stranded DNA binding coupled to a flexible C-terminal tail reminiscent of bacterial SSB that mediates interactions with other proteins. We demonstrate that Sulfolobus solfataricus SSB can melt DNA containing a mismatch or DNA lesion specifically in vitro. We suggest that a potential role for SSB in archaea is the detection of DNA damage due to local destabilisation of the DNA double helix, followed by recruitment of specific repair proteins. Proteins interacting specifically with a single-stranded DNA:SSB complex include several known or putative DNA repair proteins and DNA helicases.     

Replication and recombination of herpes simplex virus DNA

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.     

Structure of the SSB-DNA polymerase III interface and its role in DNA replication

Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.     

Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.     

Conformational changes in DNA‐binding proteins: Relationships with precomplex features and contributions to specificity and stability

Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA‐binding proteins by superimposing DNA‐bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA‐binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Proteins 2014; 82:841–857. © 2013 Wiley Periodicals, Inc.     

Replication protein A complex in Thermococcus kodakarensis interacts with DNA polymerases and helps their effective strand synthesis

Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks.     

Rtt107 is required for recruitment of the SMC5/6 complex to DNA double strand breaks

Genome integrity is maintained by a network of DNA damage response pathways, including checkpoints and DNA repair processes. In Saccharomyces cerevisiae, the BRCT domain-containing protein Rtt107/Esc4 is required for the restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. In addition to its well characterized interaction with the endonuclease Slx4, Rtt107 interacts with a number of other DNA repair and recombination proteins. These include the evolutionarily conserved SMC5/6 complex, which is involved in numerous chromosome maintenance activities, such as DNA repair, chromosome segregation, and telomere function. The interaction between Rtt107 and the SMC5/6 complex was mediated through the N-terminal BRCT domains of Rtt107 and the Nse6 subunit of SMC5/6 and was independent of methyl methane sulfonate-induced damage and Slx4. Supporting a shared function in the DNA damage response, Rtt107 was required for recruitment of SMC5/6 to DNA double strand breaks. However, this functional relationship did not extend to other types of DNA lesions such as protein-bound nicks. Interestingly, Rtt107 was phosphorylated when SMC5/6 function was compromised in the absence of DNA-damaging agents, indicating a connection beyond the DNA damage response. Genetic analyses revealed that, although a subset of Rtt107 and SMC5/6 functions was shared, these proteins also contributed independently to maintenance of genome integrity.     

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.     

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.     

Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians

The maintenance of genomic stability relies on the coordinated action of a number of cellular processes, including activation of the DNA-damage checkpoint, DNA replication, DNA repair, and telomere homeostasis. Many proteins involved in these cellular processes use different types of functional modules to regulate and execute their functions. Recent studies have revealed that many DNA-damage checkpoint and DNA repair proteins in human cells possess the oligonucleotide/oligosaccharide-binding (OB) fold domains, which are known to bind single-stranded DNA in both prokaryotes and eukaryotes. Furthermore, during the DNA damage response, the OB folds of the human checkpoint and DNA repair proteins play critical roles in DNA binding, protein complex assembly, and regulating protein–protein interactions. These findings suggest that the OB fold is an evolutionarily conserved functional module that is widely used by genome guardians. In this review, we will highlight the functions of several well-characterized or newly discovered eukaryotic OB-fold proteins in the DNA damage response.     

The puzzle of PCNA's many partners

The identification of proteins that interact with proliferating cell nuclear antigen (PCNA) has recently been a rapidly expanding field of discovery. PCNA is involved in many aspects of DNA replication and processing, forming a sliding platform that can mediate the interaction of proteins with DNA. It is striking that many proteins bind to PCNA through a small region containing a conserved motif; these include proteins involved in cell cycle regulation as well as those involved in DNA processing. Sequential and regulated binding of motif-containing proteins to PCNA may contribute to the ordering of events during DNA replication and repair. Results from bacteriophages and archaea show that the structural basis for the interaction of this motif with PCNA is extremely ancient. The analysis of how such functional motifs have been recruited to proteins in present day organisms helps us to understand how these complex systems arose from ancestral organisms.     

Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.     

The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity

DNA damage leads to activation of several mechanisms such as DNA repair and cell-cycle checkpoints. It is evident that these different cellular mechanisms have to be finely co-ordinated. Growing evidence suggests that the Rad9/Rad1/Hus1 cell-cycle checkpoint complex (9-1-1 complex), which is recruited to DNA lesion upon DNA damage, plays a major role in DNA repair. This complex has been shown to interact with and stimulate several proteins involved in long-patch base excision repair. On the other hand, the well-characterised DNA clamp-proliferating cell nuclear antigen (PCNA) also interacts with and stimulates several of these factors. In this work, we compared the effects of the 9-1-1 complex and PCNA on flap endonuclease 1 (Fen1). Our data suggest that PCNA and the 9-1-1 complex can independently bind to and activate Fen1. Finally, acetylation of Fen1 by p300-HAT abolished the stimulatory effect of the 9-1-1 complex but not that of PCNA, suggesting a possible mechanism of regulation of this important repair pathway.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.