Proliferating cell nuclear antigen: More than a clamp for DNA polymerases

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.     

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.     

Trading places: how do DNA polymerases switch during translesion DNA synthesis?

The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.     

UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability

Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.     

Polymorphism of genes encoding SOS polymerases in natural populations of Escherichia coli

High fidelity replicative DNA polymerases can be blocked during DNA replication by various base damages, which represents a potentially lethal event. Escherichia coli possesses three DNA polymerases, PolII, PolIV and PolV, that can continue replication over such lesions in template DNA, thus allowing for cell survival. Genes coding for these enzymes, polB, dinB, and umuCD respectively, belong to the stress-inducible SOS regulon. We have analyzed the patterns of nucleotide sequence variability of genes encoding for three SOS polymerases from E. coli natural isolates in order to identify the nature of selective forces that determine their evolution. The frequency of inferred inter-strain recombination events, and the frequency of synonymous and non-synonymous base substitutions within these genes do not deviate significantly from those observed for the control group composed of 2 genes coding for DNA polymerases PolI and PolIII and 10 metabolic genes. This suggests that the loci coding for SOS polymerases are subject to selective pressure for the maintenance of their function and specificity. The fact that genes coding for translesion-synthesis (TLS) polymerases, particularly dinB and umuC homologs, have been conserved during evolution and the present analysis suggest that their activity is essential for the cellular survival and fitness.     

The effect of oxidative metabolism on spontaneous Pol zeta-dependent translesion synthesis in Saccharomyces cerevisiae

DNA lesions can stall or block high-fidelity polymerases, thus inhibiting replication. To bypass such lesions, low-fidelity translesion synthesis (TLS) polymerases can be used to insert a nucleotide across from the lesion or extend from a lesion:base mispair. When DNA repair is compromised in Saccharomyces cerevisiae, spontaneous DNA lesions can lead to a novel mutational event in which a frameshift is accompanied by one or more base pair substitutions. These "complex frameshifts" are dependent upon the TLS polymerase Pol zeta, and provide a mutational signature for mutagenic Pol zeta-dependent activity. In the current study, we have found that a specific subset of the Pol zeta-dependent mutational events requires oxidative metabolism. These results suggest that translesion bypass of spontaneously oxidized DNA bases can be a significant source of mutagenesis in repair compromised cells.     

The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.     

Ubiquitin-family modifications in the replication of DNA damage

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.     

Kohamaic acid A,a novel sesterterpenic acid,inhibits activities of DNA polymerases from deuterostomes

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.     

Initiation of genetic recombination and recombination-dependent replication

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.     

In vitro replication slippage by DNA polymerases from thermophilic organisms

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.     

The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta

This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed.     

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.     

Studying DNA–protein interactions with single-molecule Förster resonance energy transfer

Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling.     

Unlocking the sugar "steric gate" of DNA polymerases

To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the 2'-hydroxyl of an incoming ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most DNA polymerases to selectively insert deoxyribonucleotides. Recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanistic consequences of ribonucleotide misincorporation on downstream events, such as the bypass of a ribonucleotide in a DNA template and the subsequent extension of the DNA lesion bypass product. These important findings are summarized in this review.     

Distribution and roles of X-family DNA polymerases in eukaryotes

Four types of DNA polymerase (Pol beta, Pol lambda, Pol mu and TdT) have been identified in eukaryotes as members of the polymerase X-family. Only vertebrates have all four types of enzyme. Plants and fungi have one or two X-family polymerases, while protostomes, such as fruit flies and nematodes, do not appear to have any. It is possible that the well-known metabolic pathways in which these enzymes are involved are restricted to the vertebrate world. The distribution of the DNA polymerases involved in DNA repair across the various biological kingdoms differs from that of the DNA polymerases involved in chromosomal DNA replication. In this review, we focus on the interesting pattern of distribution of the X-family enzymes across biological kingdoms and speculate on their roles.