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Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea.

ABSTRACT: Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s-1), but exhibits an ?1,000-fold slower turnover rate (0.06-0.17 s-1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway.

SUBMITTER: Heider MR

PROVIDER: S-EPMC5448109 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea.

Heider Margaret R MR Burkhart Brett W BW Santangelo Thomas J TJ Gardner Andrew F AF

The Journal of biological chemistry 20170403 21

Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as <i>Thermococcus</i> rely only upon RNaseH2 to initiate the pathway. Because <i>Thermococcus</i> DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ens ...[more]

PMID: 28373277

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Project description:Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

Dataset Information

Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea.

Publications

Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea.

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