Abstract:The recent discovery of a new family of ubiquitous DNA polymerases involved in translesion synthesis has shed new light onto the biochemical basis of mutagenesis. Among these polymerases, the dinB gene product (Pol IV) is involved in mutagenesis in Escherichia coli. We show here that the activity of native Pol IV is drastically modified upon interaction with the β subunit, the processivity factor of DNA Pol III. In the absence of the β subunit Pol IV is strictly distributive and no stable complex between Pol I… Show more
“…1 C and D) and rate ( Fig. 2A) of each synthesis step were generated from a large number of events; the data were in agreement with previous single-molecule experiments for Pol III (22) and bulk data for Pol IV (23). Pauses between synthesis steps were exponentially distributed, consistent with a single rate-limiting step, and we observed that increasing the concentration of Pol III from 5 to 30 nM reduced the pause length ( Fig.…”
Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.single-molecule techniques | DNA replication | DNA repair | lesion bypass | DinB
“…1 C and D) and rate ( Fig. 2A) of each synthesis step were generated from a large number of events; the data were in agreement with previous single-molecule experiments for Pol III (22) and bulk data for Pol IV (23). Pauses between synthesis steps were exponentially distributed, consistent with a single rate-limiting step, and we observed that increasing the concentration of Pol III from 5 to 30 nM reduced the pause length ( Fig.…”
Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.single-molecule techniques | DNA replication | DNA repair | lesion bypass | DinB
“…E. coli contains two Y-family polymerases, PolIV and PolV; both are DNA damage inducible and belong to the SOS regulon. PolIV has an extremely low affinity for the naked primer-template substrate and heavily relies on the β clamp (a bacterial functional homolog of PCNA) to load onto DNA [87,88]. In vitro studies indicate that PolIV and its archeal homolog Dpo4 are relatively faithful polymerases at the incorporation step and the low fidelity primarily results from poor discrimination between correct and incorrect incoming nucleotides at the binding stage and the capacity to elongate mismatched primer template, which results in -1 frameshift mutations [89,90].…”
Section: Ddt In Saccharomyces Cerevisiaementioning
npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.
DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...
“…All five E. coli DNA polymerases appear to interact with the replication processivity factor, the b-clamp (Hughes et al, 1991;Kim and McHenry, 1996;Lopez de Saro and O'Donnell, 2001;Tang et al, 2000;Wagner et al, 2000). The three SOS-inducible DNA polymerases (II, IV and V) contain a short peptide (consensus motif: QLxLF) that was identified in a yeast two-hybrid screen to mediate their interaction with the b-clamp (Dalrymple et al, 2001).…”
Section: The Emerging Complexity Of Tls Pathways In Vivomentioning
Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content. Despite the efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated. Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS). These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency. We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.
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