Topoisomerases are essential enzymes that solve topological problems arising from the double-helical structure of DNA. As a consequence, one should have naively expected to find homologous ...topoisomerases in all cellular organisms, dating back to their last common ancestor. However, as observed for other enzymes working with DNA, this is not the case. Phylogenomics analyses indicate that different sets of topoisomerases were present in the most recent common ancestors of each of the three cellular domains of life (some of them being common to two or three domains), whereas other topoisomerases families or subfamilies were acquired in a particular domain, or even a particular lineage, by horizontal gene transfers. Interestingly, two groups of viruses encode topoisomerases that are only distantly related to their cellular counterparts. To explain these observations, we suggest that topoisomerases originated in an ancestral virosphere, and that various subfamilies were later on transferred independently to different ancient cellular lineages. We also proposed that topoisomerases have played a critical role in the origin of modern genomes and in the emergence of the three cellular domains.
Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the ...coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time
in vitro
formation of the ternary XRCC1 complex with the key enzymes of SP BER — DNA polymerase β (Polβ) and DNA ligase IIIa (LiglIIa) — using the fluorescence-based technique. It was found that Polβ directly interacts with LiglIIa, but their complex is less stable than the XRCC1—Polβ and XRCC1—LigIIIa complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polβ), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.
DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before ...the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.
Display omitted
•Replication fork collapse is triggered by mitotic CDK-dependent CMG unloading•Mitotic CMG unloading requires the E3 ubiquitin ligase TRAIP and the p97 ATPase•Mitotic processing of stalled forks enables high-fidelity chromosome segregation•New model for the generation of complex chromosome rearrangements
Mitotic entry before completion of DNA replication causes genome instability via an unknown mechanism. Using Xenopus egg extracts, Deng et al. find that mitotic cyclin-dependent kinase triggers replication fork breakage and DNA rearrangements. The mechanism requires TRAIP-dependent ubiquitylation of the replicative helicase followed by p97 ATPase-dependent helicase removal from chromatin.
Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been ...developed whereby alternative hydrogen‐bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base‐pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.
Hydrogen bonding or rather more hydrophobic? Two different pairing concepts are the basis for the currently most promising unnatural base pair (UBP) candidates. This review mainly focusses on structural studies investigating UBP pairings in free duplex DNA or the active site of KlenTaq DNA polymerase, but also highlights the general mechanisms for the acceptance of these unnatural substrates by DNA polymerases.
Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ), the unique replicase inside mitochondria, plays a key role in the ...fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.
Display omitted
•How does PCNA co-ordinate Okazaki fragment synthesis, processing and ligation?•Physical and functional interaction between DNA polymerase δ and DNA ligase I.•Both DNA ligase I and ...FEN1 associate with DNA polymerase δ-PCNA during gap filling DNA synthesis.•A double-dumbbell complex of DNA ligase I and PCNA remains on the ligated DNA.•Dynamic PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.
More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5′ end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.
DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis ...(TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase η (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion.
Background: Translesion synthesis involves proliferating cell nuclear antigen (PCNA) monoubiquitination and polymerase switching.
Results: C1orf124 is required for cell survival following UV damage. It binds to monoubiquitinated PCNA and participates in polymerase switching.
Conclusion: C1orf124 serves as a central platform that facilitates translesion synthesis.
Significance: This study provides a mechanism for translesion synthesis.
DNA mismatch repair Kunkel, Thomas A; Erie, Dorothy A
Annual review of biochemistry,
01/2005, Letnik:
74, Številka:
1
Journal Article
Recenzirano
Odprti dostop
DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA ...transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.
Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than 1/105, and this precision is improved to about 1/107 by their ...proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (GO) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, GO/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of GO might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.
Background: We studied the interplay between base excision repair (BER) of 8-oxoguanine (GO) and mismatch repair (MMR).
Results: BER and MMR interact during the processing of GO/A but not GO/C mispairs.
Conclusion: BER of GO-containing lesions appears to be regulated.
Significance: BER intermediates were believed to be unavailable to other pathways of DNA metabolism. This hypothesis may be incorrect.
Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the ...mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which bidirectional replication proceeds from a replication origin. We find that the rate, the genetic control, and the types and frequencies of nucleotides inserted opposite the AP site are very similar for both the leading and the lagging DNA strands, and that an A is predominantly inserted opposite the AP site, whereas C insertion by Rev1 constitutes a much less frequent event. In striking contrast, in studies that have been reported previously for AP bypass with gapped-duplex and single-stranded plasmids, it has been shown that a C is the predominant nucleotide inserted opposite the AP site. We discuss the implications of our observations for the mechanisms of TLS on the leading versus the lagging DNA strand and suggest that lesion bypass during replication involves the coordination of activities of the replicative Pol with that of the lesion-bypass Pol.
PDF
