Dataset Information

Deletion of TLS polymerases promotes homologous recombination in Arabidopsis.

ABSTRACT: Unrepaired DNA damage hinders the maintenance of genome integrity because it blocks the catalytic activity of replicase. The stalled replication fork can be processed through either translesion synthesis (TLS) with specific polymerases, or replication using the undamaged template. To investigate how TLS activities are regulated and how the stalled replication fork is processed in plants, reversion frequencies and homologous recombination (HR) frequencies were analyzed using GUS-based substrates. The HR frequencies in plants deficient in DNA polymerase ? (Pol ?) or Rev1 were higher than that in wildtype plants under normal conditions, and were significantly increased by ultraviolet light irradiation. Heat shock protein (HSP) 90 is known to be involved in various stress responses. To examine the role of HSP90 in the regulation of damage tolerance, we analyzed reversion frequencies and HR frequencies in plants grown in the presence of a HSP inhibitor, geldanamycin (GDA). Reversion frequency was lower in GDA-treated plants than in mock-treated plants. Though the HR frequency was higher in GDA-treated wildtype plants than in mock-treated plants, no significant difference was detected in Rev1-deficient plants. In yeast, TLS polymerases interacted with each other or with a replication clump component, proliferating cell nuclear antigen (PCNA). HSP90 interacted with REV1 or REV7 in Nicotiana benthamiana cells. These results suggest that HSP90 interacts with TLS polymerase(s), which promotes error-prone TLS in plants.

SUBMITTER: Sakamoto AN

PROVIDER: S-EPMC6128680 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Deletion of TLS polymerases promotes homologous recombination in Arabidopsis.

Sakamoto A N AN Kaya H H Endo M M

Plant signaling & behavior 20180626 7

Unrepaired DNA damage hinders the maintenance of genome integrity because it blocks the catalytic activity of replicase. The stalled replication fork can be processed through either translesion synthesis (TLS) with specific polymerases, or replication using the undamaged template. To investigate how TLS activities are regulated and how the stalled replication fork is processed in plants, reversion frequencies and homologous recombination (HR) frequencies were analyzed using GUS-based substrates. ...[more]

PMID: 29944437

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Project description:Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.ImportanceCells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination, providing a new function for MutS2. We found that cells lacking mutS2 are sensitive to DNA damage that requires homologous recombination for repair and have reduced transformation efficiency. Further analysis indicates that the C-terminal Smr domain requires the N-terminal portion of MutS2 for function in vivo Moreover, we show that a mutS2 deletion is additive with a recU deletion, suggesting that these proteins have a redundant function in homologous recombination. Together, our study shows that MutS2 proteins have adapted different functions that impact recombination.

Dataset Information

Deletion of TLS polymerases promotes homologous recombination in Arabidopsis.

Publications

Deletion of TLS polymerases promotes homologous recombination in Arabidopsis.

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