scholarly journals Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1045 ◽  
Author(s):  
Gaëlle Hogrel ◽  
Yang Lu ◽  
Nicolas Alexandre ◽  
Audrey Bossé ◽  
Rémi Dulermo ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Hyperthermophilic Archaea ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Domains Of Life ◽  
D Loop ◽  
Main Protein ◽  
Stalled Replication Forks

Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.

Characterization of a dam mutant of Haemophilus influenzae Rd

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3773-3781 ◽  
Author(s):  
Piotr Zaleski ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
High Sensitivity ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Phenotypic Properties ◽  
Chloramphenicol Resistance ◽  
Double Strand Dna Breaks ◽  
Dam Methylation ◽  
Dna Strands

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for CamR transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.

scholarly journals Involvement of the essential yeast DNA polymerases in induced gene conversion.

1999 ◽  
Vol 46 (4) ◽  
pp. 862-872 ◽  
Author(s):  
A Hałas ◽  
A Ciesielski ◽  
J Zuk
Keyword(s):  
Dna Synthesis ◽  
Gene Conversion ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Alpha ◽  
Genes Encoding ◽  
Mitotic Gene Conversion ◽  
Dna Strand

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.

scholarly journals Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

2021 ◽  
Author(s):  
Paul Kaminski ◽  
Shiyuan Hong ◽  
Takeyuki Kono ◽  
Paul Hoover ◽  
Laimonis A. Laimins
Keyword(s):  
Dna Cleavage ◽  
Genome Replication ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Damage Repair ◽  
High Risk Hpv ◽  
Strand Passage ◽  
Covalent Intermediate ◽  
Do So

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2b is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2b. Upon DNA cleavage a covalent intermediate DNA-TOP2b (TOP2bcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2bcc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2b proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2b and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2b with shRNAs reduced DNA breaks by over 50% as determined through COMET assays.  Furthermore this correlated with substantially reduced formation of repair foci such as gH2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2b also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2b. This study identifies TOP2b as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

scholarly journals 53BP1 and BRCA1 control pathway choice for stalled replication restart

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yixi Xu ◽  
Shaokai Ning ◽  
Zheng Wei ◽  
Ran Xu ◽  
Xinlin Xu ◽  
...  
Keyword(s):  
Genome Stability ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Independent Manner ◽  
Double Strand Dna Breaks ◽  
Replication Restart ◽  
Tumor Prevention ◽  
Pathway Choice ◽  
Break Induced Replication ◽  
Stalled Replication Forks

The cellular pathways that restart stalled replication forks are essential for genome stability and tumor prevention. However, how many of these pathways exist in cells and how these pathways are selectively activated remain unclear. Here, we describe two major fork restart pathways, and demonstrate that their selection is governed by 53BP1 and BRCA1, which are known to control the pathway choice to repair double-strand DNA breaks (DSBs). Specifically, 53BP1 promotes a fork cleavage-free pathway, whereas BRCA1 facilitates a break-induced replication (BIR) pathway coupled with SLX-MUS complex-mediated fork cleavage. The defect in the first pathway, but not DSB repair, in a 53BP1 mutant is largely corrected by disrupting BRCA1, and vice versa. Moreover, PLK1 temporally regulates the switch of these two pathways through enhancing the assembly of the SLX-MUS complex. Our results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a DSB repair-independent manner.

scholarly journals Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Zhiguo Zhang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Genome Duplication ◽  
Dna Polymerases ◽  
Distinct Pattern ◽  
Replication Forks ◽  
Okazaki Fragments ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Stalled Replication Forks

ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal.

scholarly journals Role of 53BP1 in end protection and DNA synthesis at DNA breaks

2021 ◽  
Author(s):  
Jacob Paiano ◽  
Nicholas Zolnerowich ◽  
Wei Wu ◽  
Raphael Pavani ◽  
Chen Wang ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Nuclease Activity ◽  
Extent Of Resection ◽  
Strand Break ◽  
Dna Breaks ◽  
Initial Processing ◽  
The Relationship ◽  
Dna Ends ◽  
Insight Into

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

scholarly journals The shrinking arterial genome: role of double strand DNA breaks in age‐related DNA deletion (912.9)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Richard Morgan ◽  
Stephen Ives ◽  
Richard Cawthon ◽  
Robert Andtbacka ◽  
Dirk Noyes ◽  
...  
Keyword(s):  
Dna Breaks ◽  
Dna Deletion ◽  
Double Strand Dna Breaks ◽  
Age Related ◽  
Double Strand Dna
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