scholarly journals A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

2019 ◽  
Vol 116 (51) ◽  
pp. 25591-25601 ◽  
Author(s):  
Seungwoo Chang ◽  
Karel Naiman ◽  
Elizabeth S. Thrall ◽  
James E. Kath ◽  
Slobodan Jergic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Genome Stability ◽  
Structural Changes ◽  
Translesion Synthesis ◽  
Dynamic Interaction ◽  
Dna Lesions ◽  
Exonuclease Activity ◽  
Replication Fidelity ◽  
Proper Resolution

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within theEscherichia colireplisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

scholarly journals A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

2019 ◽  
Author(s):  
Seungwoo Chang ◽  
Karel Naiman ◽  
Elizabeth S. Thrall ◽  
James E. Kath ◽  
Slobodan Jergic ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Genome Stability ◽  
Structural Changes ◽  
Translesion Synthesis ◽  
Dynamic Interaction ◽  
Dna Lesions ◽  
Exonuclease Activity ◽  
Replication Fidelity ◽  
Single Stranded Dna ◽  
Proper Resolution

AbstractDNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

scholarly journals Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ

2015 ◽  
Vol 29 (24) ◽  
pp. 2588-2602 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Jeseong Park ◽  
Juan Conde ◽  
Maki Wakamiya ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Crucial Role ◽  
Genome Stability ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Dna Lesions ◽  
Human Cells ◽  
Uv Lesions ◽  
Human And Mouse

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

scholarly journals DNA translocation mechanism of an XPD family helicase

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Kaiying Cheng ◽  
Dale B Wigley
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Genome Stability ◽  
Family Members ◽  
Dna Lesions ◽  
Human Diseases ◽  
Dna Translocation ◽  
Conserved Residues ◽  
Translocation Mechanism ◽  
Bulky Dna Lesions

The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily two helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5’−3’ direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5’−3’ translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesions might stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.

Structural changes in 16S RNA from Escherichia coli upon unfolding by urea

Biopolymers ◽  
1993 ◽  
Vol 33 (11) ◽  
pp. 1747-1755 ◽  
Author(s):  
A. A. Timchenko ◽  
J. Langowski ◽  
I. N. Serdyuk
Keyword(s):  
Escherichia Coli ◽  
Structural Changes ◽  
16S Rna

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

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.

scholarly journals DNA–protein crosslink proteases in genome stability

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Annamaria Ruggiano ◽  
Kristijan Ramadan
Keyword(s):  
Dna Replication ◽  
Cancer Therapy ◽  
Genome Stability ◽  
Dna Lesions ◽  
Protein Component ◽  
Human Diseases ◽  
Direct Link ◽  
The Past ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

AbstractProteins covalently attached to DNA, also known as DNA–protein crosslinks (DPCs), are common and bulky DNA lesions that interfere with DNA replication, repair, transcription and recombination. Research in the past several years indicates that cells possess dedicated enzymes, known as DPC proteases, which digest the protein component of a DPC. Interestingly, DPC proteases also play a role in proteolysis beside DPC repair, such as in degrading excess histones during DNA replication or controlling DNA replication checkpoints. Here, we discuss the importance of DPC proteases in DNA replication, genome stability and their direct link to human diseases and cancer therapy.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

scholarly journals The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

2016 ◽  
Vol 27 (24) ◽  
pp. 3791-3799 ◽  
Author(s):  
Hafida Sellou ◽  
Théo Lebeaupin ◽  
Catherine Chapuis ◽  
Rebecca Smith ◽  
Anna Hegele ◽  
...  
Keyword(s):  
Dna Damage ◽  
Structural Changes ◽  
Cellular Responses ◽  
Dna Lesions ◽  
Fast Kinetics ◽  
Important Player ◽  
Chromatin Relaxation ◽  
Kinetics Of ◽  
Laser Microirradiation

Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.

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