Cyclin Proteolysis and CDK Inhibitors: Two Redundant Pathways to Maintain Genome Stability in Mammalian Cells

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

Nature Communications ◽

10.1038/s41467-021-23535-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katerina Zabrady ◽

Matej Zabrady ◽

Peter Kolesar ◽

Arthur W. H. Li ◽

Aidan J. Doherty

Keyword(s):

Dna Repair ◽

Dna Synthesis ◽

Genome Stability ◽

Dna Polymerases ◽

Acquired Immunity ◽

Strand Displacement ◽

Genetic Studies ◽

Type Iiia ◽

Maintain Genome Stability ◽

Cas Proteins

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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The Protective Role of Dormant Origins in Response to Replicative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms19113569 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3569 ◽

Cited By ~ 11

Author(s):

Lilas Courtot ◽

Jean-Sébastien Hoffmann ◽

Valérie Bergoglio

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Genome Stability ◽

Replication Timing ◽

Protective Role ◽

Entire Genome ◽

Replicative Stress ◽

A Cell ◽

The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

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Control of the DNA Methylation System Component MBD2 by Protein Arginine Methylation

Molecular and Cellular Biology ◽

10.1128/mcb.00473-06 ◽

2006 ◽

Vol 26 (19) ◽

pp. 7224-7235 ◽

Cited By ~ 41

Author(s):

Choon Ping Tan ◽

Sara Nakielny

Keyword(s):

Dna Methylation ◽

Complex Formation ◽

Histone Deacetylases ◽

Mammalian Cells ◽

Genome Stability ◽

Epigenetic Modification ◽

Arginine Methylation ◽

System Component ◽

Transcription Repression ◽

Mbd Proteins

ABSTRACT DNA methylation is vital for proper chromatin structure and function in mammalian cells. Genetic removal of the enzymes that catalyze DNA methylation results in defective imprinting, transposon silencing, X chromosome dosage compensation, and genome stability. This epigenetic modification is interpreted by methyl-DNA binding domain (MBD) proteins. MBD proteins respond to methylated DNA by recruiting histone deacetylases (HDAC) and other transcription repression factors to the chromatin. The MBD2 protein is dispensable for animal viability, but it is implicated in the genesis of colon tumors. Here we report that the MBD2 protein is controlled by arginine methylation. We identify the protein arginine methyltransferase enzymes that catalyze this modification and show that arginine methylation inhibits the function of MBD2. Arginine methylation of MBD2 reduces MBD2-methyl-DNA complex formation, reduces MBD2-HDAC repression complex formation, and impairs the transcription repression function of MBD2 in cells. Our report provides a molecular description of a potential regulatory mechanism for an MBD protein family member. It is the first to demonstrate that protein arginine methyltransferases participate in the DNA methylation system of chromatin control.

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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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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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