Mechanism of strand displacement synthesis by DNA replicative polymerases

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.     

A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases

Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.     

Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases

The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active-site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain alpha-helices by 60 degrees upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40 degrees and the thumb domain re-orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non-template strand from the template strand.     

DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5'-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase beta (Pol beta) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol beta activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.     

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.     

The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.     

Sequencing double-stranded DNA by strand displacement.

Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequenced without subcloning, purification or denaturation.     

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

Damage repair DNA polymerases Y

The newly found Y-family DNA polymerases are characterized by low fidelity replication using an undamaged template and the ability to carry out translesion DNA synthesis. The crystal structures of three Y-family polymerases, alone or complexed with DNA and nucleotide substrate, reveal a conventional right-hand-like catalytic core consisting of finger, thumb and palm domains. The finger and thumb domains are unusually small resulting in an open and spacious active site, which can accommodate mismatched base pairs as well as various DNA lesions. Although devoid of a 3'-->5' exonuclease activity, the Y-family polymerases possess a unique "little finger" domain that facilitates DNA association, catalytic efficiency and interactions with auxiliary factors. Expression of Y-family polymerases is often induced by DNA damage, and their recruitment to the replication fork is mediated by beta-clamp, clamp loader, single-strand-DNA-binding protein and RecA in Escherichia coli, and by ubiquitin-modified proliferating cell nuclear antigen in yeast.     

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight (~1.17 MDa) species, is a DNA-dependent ATPase, has 3′→5′ DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.     

Structural basis for uracil recognition by archaeal family B DNA polymerases

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C-->A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.     

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair

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Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.     

Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.     

Hijacked DNA repair proteins and unchained DNA polymerases

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2–MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases η, ι and θ in synthesizing mutations, and conclude that polymerase η is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.     

Modeling translocation dynamics of strand displacement DNA synthesis by DNA polymerase I

A model is presented for the translocation dynamics of the strand displacement DNA synthesis by DNA polymerases such as polymerase I family. (i) The model gives an explanation to the experimental results which showed that the rate of strand displacement DNA synthesis is nearly consistent with that of single stranded primer extension synthesis, although the two are expected to have substantial differences in their energetics. (ii) During strand displacement DNA synthesis, the pausing at the specific sequence is considered to be due to an affinity of the fingers subdomain for the specific sequence of dsDNA downstream of the single strand. The theoretical results on the sequence-dependent pausing dynamics such as the mean pausing lifetimes and the distribution of the pausing lifetime are consistent with the experimental data. Moreover, predicted results are presented for the binding affinity of the fingers subdomain for the specific sequence of dsDNA and the dependence of the mean sequence-dependent pausing lifetime on the external force acting on the polymerase.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

Structure and function of a mycobacterial NHEJ DNA repair polymerase

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown.