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Group II intron-like reverse transcriptases function in double-strand break repair.

ABSTRACT: Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. We found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase θ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ). We also found that a group II intron RT can function similarly in DNA repair, with reciprocal active-site substitutions showing isoleucine favors MMEJ and alanine favors primer extension in both enzymes. These DNA repair functions utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have inherent ability to function in DSBR in a wide range of organisms.

SUBMITTER: Park SK

PROVIDER: S-EPMC9530004 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Group II intron-like reverse transcriptases function in double-strand break repair.

Park Seung Kuk SK Mohr Georg G Yao Jun J Russell Rick R Lambowitz Alan M AM

Cell 20220915 20

Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. We found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase θ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) ...[more]

PMID: 36113466

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Project description:BackgroundDNA double strand break (DSB) repair enzymes are thought to be necessary for retroviral infection, especially for the post-integration repair and circularization of viral cDNA. However, the detailed roles of DSB repair enzymes in retroviral infection remain to be elucidated.ResultsA GFP reporter assay showed that the infectivity of an HIV-based vector decreased in ATM- and DNA-PKcs-deficient cells when compared with their complemented cells, while that of an MLV-based vector was diminished in Mre11- and DNA-PKcs-deficient cells. By using a method based on inverse- and Alu-PCR, we analyzed sequences around 3' HIV-1 integration sites in ATM-, Mre11- and NBS1- deficient cells. Increased abnormal junctions between the HIV-1 provirus and the host DNA were found in these mutant cell lines compared to the complemented cell lines and control MRC5SV cells. The abnormal junctions contained two types of insertions: 1) GT dinucleotides, which are normally removed by integrase during integration, and 2) inserted nucleotides of unknown origin. Artemis-deficient cells also showed such abnormalities. In Mre11-deficient cells, part of a primer binding site sequence was also detected. The 5' host-virus junctions in the mutant cells also contained these types of abnormal nucleotides. Moreover, the host-virus junctions of the MLV provirus showed similar abnormalities. These findings suggest that DSB repair enzymes play roles in the 3'-processing reaction and protection of the ends of viral DNA after reverse transcription. We also identified both 5' and 3' junctional sequences of the same provirus by inverse PCR and found that only the 3' junctions were abnormal with aberrant short repeats, indicating that the integration step was partially impaired in these cells. Furthermore, the conserved base preferences around HIV-1 integration sites were partially altered in ATM-deficient cells.ConclusionsThese results suggest that DSB repair enzymes are involved in multiple steps including integration and pre-integration steps during retroviral replication.

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Group II intron-like reverse transcriptases function in double-strand break repair.

Publications

Group II intron-like reverse transcriptases function in double-strand break repair.

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