Cargando…

Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication...

Descripción completa

Detalles Bibliográficos
Autores principales: Vanoli, Fabio, Fumasoni, Marco, Szakal, Barnabas, Maloisel, Laurent, Branzei, Dana
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978687/
https://www.ncbi.nlm.nih.gov/pubmed/21085632
http://dx.doi.org/10.1371/journal.pgen.1001205
_version_ 1782191282553094144
author Vanoli, Fabio
Fumasoni, Marco
Szakal, Barnabas
Maloisel, Laurent
Branzei, Dana
author_facet Vanoli, Fabio
Fumasoni, Marco
Szakal, Barnabas
Maloisel, Laurent
Branzei, Dana
author_sort Vanoli, Fabio
collection PubMed
description Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.
format Text
id pubmed-2978687
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-29786872010-11-17 Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch Vanoli, Fabio Fumasoni, Marco Szakal, Barnabas Maloisel, Laurent Branzei, Dana PLoS Genet Research Article Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair. Public Library of Science 2010-11-11 /pmc/articles/PMC2978687/ /pubmed/21085632 http://dx.doi.org/10.1371/journal.pgen.1001205 Text en Vanoli et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Vanoli, Fabio
Fumasoni, Marco
Szakal, Barnabas
Maloisel, Laurent
Branzei, Dana
Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title_full Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title_fullStr Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title_full_unstemmed Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title_short Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch
title_sort replication and recombination factors contributing to recombination-dependent bypass of dna lesions by template switch
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978687/
https://www.ncbi.nlm.nih.gov/pubmed/21085632
http://dx.doi.org/10.1371/journal.pgen.1001205
work_keys_str_mv AT vanolifabio replicationandrecombinationfactorscontributingtorecombinationdependentbypassofdnalesionsbytemplateswitch
AT fumasonimarco replicationandrecombinationfactorscontributingtorecombinationdependentbypassofdnalesionsbytemplateswitch
AT szakalbarnabas replicationandrecombinationfactorscontributingtorecombinationdependentbypassofdnalesionsbytemplateswitch
AT maloisellaurent replicationandrecombinationfactorscontributingtorecombinationdependentbypassofdnalesionsbytemplateswitch
AT branzeidana replicationandrecombinationfactorscontributingtorecombinationdependentbypassofdnalesionsbytemplateswitch