Histone H2AX Is Involved in FoxO3a-Mediated Transcriptional Responses to Ionizing Radiation to Maintain Genome Stability

AbstractCRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

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Phosphorylated Histone H2AX Foci Persist on Rejoined Mitotic Chromosomes in Normal Human Diploid Cells Exposed to Ionizing Radiation

Radiation Research ◽

10.1667/rr3508.1 ◽

2006 ◽

Vol 165 (3) ◽

pp. 269-276 ◽

Cited By ~ 57

Author(s):

Masatoshi Suzuki ◽

Keiji Suzuki ◽

Seiji Kodama ◽

Masami Watanabe

Keyword(s):

Ionizing Radiation ◽

Mitotic Chromosomes ◽

Histone H2ax ◽

Normal Human ◽

Human Diploid Cells ◽

Diploid Cells

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Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Journal of Cell Biology ◽

10.1083/jcb.200905006 ◽

2009 ◽

Vol 187 (1) ◽

pp. 15-23 ◽

Cited By ~ 52

Author(s):

Kevin D. Smith ◽

Michael A. Fu ◽

Eric J. Brown

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nucleotide Incorporation ◽

Pathway Activation ◽

Maintain Genome Stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

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Transcriptional responses to ultraviolet and ionizing radiation: An approach based on graph curvature

2016 IEEE International Conference on Bioinformatics and Biomedicine (BIBM) ◽

10.1109/bibm.2016.7822706 ◽

2016 ◽

Author(s):

Yongxin Chen ◽

Jung Hun Oh ◽

Romeil Sandhu ◽

Sangkyu Lee ◽

Joseph O. Deasy ◽

...

Keyword(s):

Ionizing Radiation ◽

Transcriptional Responses

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Author Correction: ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability

Nature Communications ◽

10.1038/s41467-018-04599-6 ◽

2018 ◽

Vol 9 (1) ◽

Author(s):

Jung-Hee Lee ◽

Seon-Joo Park ◽

Gurusamy Hariharasudhan ◽

Min-Ji Kim ◽

Sung Mi Jung ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Genome Stability ◽

Damage Response ◽

Maintain Genome Stability

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Replisome bypass of a protein-based R-loop block by Pif1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020189117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30354-30361

Author(s):

Grant D. Schauer ◽

Lisanne M. Spenkelink ◽

Jacob S. Lewis ◽

Olga Yurieva ◽

Stefan H. Mueller ◽

...

Keyword(s):

Single Molecule ◽

Genome Stability ◽

Genome Instability ◽

Multiprotein Complex ◽

Genome Maintenance ◽

Visualization Technique ◽

General Displacement ◽

Maintain Genome Stability ◽

Protein Blocks

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstitutedSaccharomyces cerevisiaereplisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

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Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa054 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3053-3070

Author(s):

Esther C Morafraile ◽

Alberto Bugallo ◽

Raquel Carreira ◽

María Fernández ◽

Cristina Martín-Castellanos ◽

...

Keyword(s):

Genome Stability ◽

Nuclease Activity ◽

S Phase ◽

Exonuclease Activity ◽

Replication Forks ◽

Post Translational Modifications ◽

Replicative Stress ◽

Stalled Replication Forks ◽

Maintain Genome Stability ◽

Specific Contribution

Abstract The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

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Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

Science Advances ◽

10.1126/sciadv.aay4453 ◽

2020 ◽

Vol 6 (7) ◽

pp. eaay4453

Author(s):

A. Castañeda-García ◽

I. Martín-Blecua ◽

E. Cebrián-Sastre ◽

A. Chiner-Oms ◽

M. Torres-Puente ◽

...

Keyword(s):

Mismatch Repair ◽

Mycobacterium Smegmatis ◽

Genome Stability ◽

Structural Homology ◽

Spontaneous Mutations ◽

Wild Type ◽

Key Factor ◽

Maintain Genome Stability ◽

Mmr Proteins

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

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