Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.     

Stimulation of Oligonucleotide-Directed Gene Correction by Redβ Expression and MSH2 Depletion in Human HT1080 Cells

The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redβ, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.     

The ring-type polymerase sliding clamp family

Ring-type polymerases consist of a DNA polymerase, a ring-shaped sliding clamp protein and a clamp-loading complex. Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and the β clamp in prokaryotes. Both PCNA and β form a ring around DNA, which is made up of two subunits of three domains each in β but three subunits of two domains each in PCNA. Despite this difference and a lack of detectable sequence homology, the structures of the two rings are very similar. The sliding clamp slides along DNA and tethers the polymerase to the DNA, enabling rapid and processive DNA replication.     

Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states

Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redβ forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redβ in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redβ forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redβ in cells active for recombination and find it to range from 7 to 27 μM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.     

Classification and evolutionary history of the single-strand annealing proteins,RecT, Redβ, ERF and RAD52

Background  

The DNA single-strand annealing proteins (SSAPs), such as RecT, Redβ, ERF and Rad52, function in RecA-dependent and RecA-independent DNA recombination pathways. Recently, they have been shown to form similar helical quaternary superstructures. However, despite the functional similarities between these diverse SSAPs, their actual evolutionary affinities are poorly understood.     

Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (β^C) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β⁺), and one β^C subunit (β⁺/β^C). The β⁺/β^C heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β⁺. Moreover, β⁺/β^C interacted normally with Pol III, and stimulated replication to the same extent as did β⁺. Finally, β⁺/β^C was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.     

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.     

Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine

The prokaryotic DNA polymerase III clamp loader complex loads the β clamp onto DNA to link the replication complex to DNA during processive synthesis and unloads it again once synthesis is complete. This minimal complex consists of one δ, one δ′ and three γ subunits, all of which possess an AAA+ module—though only the γ subunit exhibits ATPase activity. Here clues to underlying clamp loader mechanisms are obtained through Bayesian inference of various categories of selective constraints imposed on the γ and δ′ subunits. It is proposed that a conserved histidine is ionized via electron transfer involving structurally adjacent residues within the sensor 1 region of γ's AAA+ module. The resultant positive charge on this histidine inhibits ATPase activity by drawing the negatively charged catalytic base away from the active site. It is also proposed that this arrangement is disrupted upon interaction of DNA with basic residues in γ implicated previously in DNA binding, regarding which a lysine that is near the sensor 1 region and that is highly conserved both in bacterial and in eukaryotic clamp loader ATPases appears to play a critical role. γ ATPases also appear to utilize a trans-acting threonine that is donated by helix 6 of an adjacent γ or δ′ subunit and that assists in the activation of a water molecule for nucleophilic attack on the γ phosphorous atom of ATP. As eukaryotic and archaeal clamp loaders lack most of these key residues, it appears that eubacteria utilize a fundamentally different mechanism for clamp loader activation than do these other organisms.     

Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly

The Escherichia coli clamp loader, γ complex (γ₃δδ′λψ), catalyzes ATP-driven assembly of β clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which γ complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by γ complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between γ complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence—HRVW₂₇₉QNRR—in δ subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of δ-W279 weakens γ complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (δ-R277, δ-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

Dissimilar mispair-recognition spectra of Arabidopsis DNA-mismatch-repair proteins MSH2*MSH6 (MutSalpha) and MSH2*MSH7 (MutSgamma)

Besides orthologs of other eukaryotic mismatch-repair (MMR) proteins, plants encode MSH7, a paralog of MSH6. The Arabidopsis thaliana recognition heterodimers AtMSH2·MSH6 (AtMutSα) and AtMSH2·MSH3 (AtMutSβ) were previously found to bind the same subsets of mismatches as their counterparts in other eukaryotes—respectively, base–base mismatches and single extra nucleotides, loopouts of extra nucleotides (one or more) only—but AtMSH2·MSH7 (AtMutSγ) bound well only to a G/T mismatch. To test hypotheses that MSH7 might be specialized for G/T, or for base mismatches in 5-methylcytosine contexts, we compared binding of AtMutSα and AtMutSγ to a series of mismatched DNA oligoduplexes, relative to their (roughly similar) binding to G/T DNA. AtMutSγ bound G/G, G/A, A/A and especially C/A mispairs as well or better than G/T, in contrast to MutSα, for which G/T was clearly the best base mismatch. The presence of 5-methylcytosine adjacent to or in a mispair generally lowered binding by both heterodimers, with no systematic difference between the two. Alignment of protein sequences reveals the absence in MSH7 of the clamp domains that in bacterial MutS proteins—and by inference MSH6 proteins—non-specifically bind the backbone of mismatched DNA, raising new questions as to how clamp domains enhance mismatch recogni tion. Plants must rigorously suppress mutation during mitotic division of meristematic cells that eventually give rise to gametes and may also use MMR proteins to antagonize homeologous recombination. The MSH6 versus MSH7 divergence may reflect specializations for particular mismatches and/or sequence contexts, so as to increase both DNA-replication and meiotic-recombination fidelity, or dedication of MSH6 to the former and MSH7 to the latter, consistent with genetic evidence from wheat.     

p53 and p73 display common and distinct requirements for sequence specific binding to DNA

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73β proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73β than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73β but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73β than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Δ30) demonstrates the same sequence discrimination as does p73β. In vivo, both p53 and p73β exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.     

RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase delta

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ~10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.     

Gap-Directed Translesion DNA Synthesis of an Abasic Site on Circular DNA Templates by a Human Replication Complex

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.     

Evolutionary clues to DNA polymerase III beta clamp structural mechanisms

The prokaryotic DNA polymerase III β homodimeric clamp links the replication complex to DNA during polynucleotide synthesis. This clamp is loaded onto DNA and unloaded by the clamp loader complex, the δ subunit of which by itself can bind to and open the clamp. β Clamps from diverse bacteria were examined using contrast hierarchical alignment and interaction network (CHAIN) analysis, a statistical approach that categorizes and measures the evolutionary constraints imposed on protein sequences by natural selection. Some constraints are subtle inasmuch as they are unique to certain bacteria. Examination of corresponding molecular interactions within structures of the Escherichia coli β dimeric and δ–β complexes reveals that N320, Y323 and R176, which are subject to very strong constraints, form a substructure that may serve as a platform for leveraging and directing δ-induced conformational changes. N320 may play a prominent role, as it is strategically situated between this substructure and regions linked to δ binding and opening of β’s dimeric interface. R176 appears to act as a relay between the δ binding site and the clamp’s central hole. Other residues subject to strong constraints are likewise associated with structurally important features. For example, two pairs of interacting residues, R269/E304 and K74/E300, form salt bridges at the dimeric interface, while the C-terminal residues M362, P363, M364 and R365 appear to play key roles in δ binding. Q149 and K198 appear to sense DNA within the clamp’s central hole while other residues may relay this information to the δ binding site. Mutagenesis experiments designed to explore possible mechanisms are proposed.     

The sliding clamp tethers the endonuclease domain of MutL to DNA

The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (β-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-β interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-β complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-β interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein.     

Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

Amino Acid Sequence Analysis of Reverse Transcriptase Subunits from Avian Myeloblastosis Virus

The NH₂-terminal amino acid sequences of the α and β chains of avian myeloblastosis αβ DNA polymerase were determined by using microsequence analysis in the subnanomole range and were found to be identical up to 17 residues. The common sequence was as follows: Thr-Val-Ala-Leu-His-Leu-Ala-Ile-Pro-Leu-Lys-Trp-Lys-Pro-Asn-His-Thr-. This result provides convincing chemical evidence that the α chain is derived from the NH₂-terminal region of the β chain by proteolytic cleavage, whereas the amino acid composition for these α and β subunits and p32 DNA endonuclease suggests that the latter is derived from the carboxyl-terminal region of the β chain.     

A new recombineering system for Photorhabdus and Xenorhabdus

Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.