Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

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Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

10.1101/2021.02.15.431208 ◽

2021 ◽

Author(s):

Asha Mary Joseph ◽

Saheli Daw ◽

Ismath Sadhir ◽

Anjana Badrinarayanan

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Replication Fork ◽

Excision Repair ◽

Genome Integrity ◽

Replication Machinery ◽

Gap Filling ◽

Independent Manner ◽

Dna Lesion ◽

Nucleotide Excision

AbstractTranslesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of the polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in its absence. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

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The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

Nucleic Acids Research ◽

10.1093/nar/27.16.3276 ◽

1999 ◽

Vol 27 (16) ◽

pp. 3276-3282 ◽

Cited By ~ 65

Author(s):

P. P. H. Van Sloun ◽

J. G. Jansen ◽

G. Weeda ◽

L. H. F. Mullenders ◽

A. A. van Zeeland ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Embryonic Stem ◽

Genotoxic Effects ◽

Nucleotide Excision

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Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00308-10 ◽

2010 ◽

Vol 30 (13) ◽

pp. 3206-3215 ◽

Cited By ~ 45

Author(s):

Nayun Kim ◽

Sue Jinks-Robertson

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Genome Integrity ◽

Genetic Studies ◽

Abasic Sites ◽

Dna And Rna ◽

Nucleotide Excision ◽

Ap Sites ◽

Base Excision ◽

Ap Site

ABSTRACT Abasic (AP) sites are potent blocks to DNA and RNA polymerases, and their repair is essential for maintaining genome integrity. Although AP sites are efficiently dealt with through the base excision repair (BER) pathway, genetic studies suggest that repair also can occur via nucleotide excision repair (NER). The involvement of NER in AP-site removal has been puzzling, however, as this pathway is thought to target only bulky lesions. Here, we examine the repair of AP sites generated when uracil is removed from a highly transcribed gene in yeast. Because uracil is incorporated instead of thymine under these conditions, the position of the resulting AP site is known. Results demonstrate that only AP sites on the transcribed strand are efficient substrates for NER, suggesting the recruitment of the NER machinery by an AP-blocked RNA polymerase. Such transcription-coupled NER of AP sites may explain previously suggested links between the BER pathway and transcription.

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Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Nature ◽

10.1038/nature08076 ◽

2009 ◽

Vol 459 (7248) ◽

pp. 808-813 ◽

Cited By ~ 84

Author(s):

Julie L. Tubbs ◽

Vitaly Latypov ◽

Sreenivas Kanugula ◽

Amna Butt ◽

Manana Melikishvili ◽

...

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Nucleotide Excision

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Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Cell Cycle ◽

10.4161/15384101.2014.973740 ◽

2015 ◽

Vol 14 (7) ◽

pp. 1103-1115 ◽

Cited By ~ 17

Author(s):

Chunhua Han ◽

Gulzar Wani ◽

Ran Zhao ◽

Jiang Qian ◽

Nidhi Sharma ◽

...

Keyword(s):

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Gap Filling ◽

Nucleotide Excision

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Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽

10.1128/msphere.00062-20 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Jia Feng ◽

Shuangyan Yao ◽

Yansong Dong ◽

Jing Hu ◽

Malcolm Whiteway ◽

...

Keyword(s):

Dna Damage ◽

Candida Albicans ◽

Nucleotide Excision Repair ◽

Dna Damage Response ◽

Excision Repair ◽

Content Type ◽

Virulence Regulation ◽

Nucleotide Excision ◽

Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

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