Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks

Chakraborty, Anirban; Tapryal, Nisha; Islam, Azharul; Sarker, Altaf H.; Manohar, Kodavati; Mitra, Joy; Hegde, Muralidhar L.; Hazra, Tapas K.

doi:10.1016/j.jbc.2023.102991

Cited by 16 publications

(23 citation statements)

References 83 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We previously described that DNA repair in mammalian brains involves dynamic multi-protein complexes 19, 20, 21, 27 . The significant discovery underlying the present study is our unexpected finding (by 2D gel and MS analysis) of an allosteric modulator of glycolysis, PFKFB3 in the TC-NHEJ complex ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington’s disease

Chakraborty,

Sreenivasmurthy,

Miller

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

We previously reported that the loss of activity of an essential DNA repair enzyme, polynucleotide kinase 3-phosphatase (PNKP), resulted in accumulation of double strand breaks (DSB) in patients brain genome in Huntingtons disease (HD) and Spinocerebellar ataxia type 3 (SCA3). Here we document that PNKP interacts with the nuclear isoform of phosphofructokinase fructose-2,6-bisphosphatase 3 (PFKFB3), which converts fructose-6-phosphate (F6P) into fructose-2,6-bisphosphate (F2,6BP), a potent allosteric modulator of glycolysis. Depletion of PFKFB3 markedly abrogates PNKP activity, thereby affecting PNKP mediated transcription-coupled non-homologous end joining (TC-NHEJ). Both PFKFB3 and F2,6BP levels are significantly lower in the nuclear extracts of HD and SCA3 patients brains. Exogenous F2,6BP restored PNKP activity in the brain nuclear extracts of those samples. Moreover, delivery of F2,6BP into HD mouse striata-derived neuronal cells restored PNKP activity, transcribed genome integrity and cellular viability. We thus postulate that F2,6BP serves in vivo as a cofactor for proper functionality of PNKP and thereby of brain health. Our results thus provide a compelling rationale for exploring therapeutic use of F2,6BP and related compounds for treating polyQ diseases.

show abstract

Section: Resultsmentioning

confidence: 99%

“…5A ) . Consequently, TC-NHEJ repair was also impaired, as evident in widespread accumulation of γH2AX and p53BP1 (both markers of DSB accumulation and p53BP1, a specific marker of C-NHEJ 19,20 ) ( Supplementary Fig. 5A , lanes 7 vs. 1) .…”

Section: Resultsmentioning

confidence: 99%

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington’s disease

Chakraborty,

Sreenivasmurthy,

Miller

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Notably, AcK226 PNKP immunocomplex showed the presence of DNA polymerase eta (Pol η). We recently provided evidence that Pol η plays a critical role in copying the sequence information via its reverse transcriptase activity from nascent RNA into DNA at the DSB sites ( 22 ). Thus, we postulate a role of Ac-K226 PNKP in the recruitment of Pol η for RNA-templated error-free repair of DSBs via the TC-NHEJ pathway.…”

Section: Discussionmentioning

confidence: 99%

“…The antibodies (Abs) used in this study and their sources are listed in Table 1 . WB analysis was performed following the protocol as described earlier ( 20 , 22 , 27 , 28 ). All primary Abs were diluted 1:500 in 5% skim milk containing TBST (1× Tris buffered saline with 1% Tween 20) buffer except GAPDH which was diluted to 1:7000.…”

Section: Methodsmentioning

confidence: 99%

Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways

Islam,

Chakraborty,

Sarker

et al. 2024

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Mammalian polynucleotide kinase 3′-phosphatase (PNKP), a DNA end-processing enzyme with 3′-phosphatase and 5′-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

show abstract

“…Rad52 is required for RNA-mediated DNA repair by facilitating pairing between homologous RNA and DNA through an inverse strand exchange activity 18,21 . The translesion DNA polymerase θ (Polθ ) that participates in alternativeend joining repair (also called microhomology-mediated end-joining or theta-mediated end joining) 22,23 , exhibits high fidelity as a reverse transcriptase (RT) on RNA-DNA templates and can stimulate repair of model DSB substrates with an RNA repair template in HEK293 cells 24 ; similar RNA-repair functions are found for Polν and Polη 25,26 . These observations suggest that RNA could act as an efficient template for the repair of targeted DSBs through recruitment of endogenous repair functions if an RNA repair --CAGAGCAGGGTCCCGCTTCCCTAAGGCCCTGCTCTGGGCTTCTGGGTTTGAGTCC--------GTCTCGTCCCAGGGCGAAGGGATTCCGGGACGAGACCCGAAGACCCAAACTCAGG------CCCUGCUCUGGGCUUCUGGGU I-TevI site…”

Section: Introductionmentioning

confidence: 99%

Harnessing RNA-based DNA repair pathways for targeted gene editing

Huynh,

Kwon,

McMurrough

et al. 2024

Preprint

View full text Add to dashboard Cite

Recent studies have revealed a role for RNA in the repair of DNA double-strand breaks. Here, we show that the asymmetric DNA overhangs generated by the small TevSaCas9 dual nuclease informs a simple and robust RNA-based editing strategy in human cells whereby Polθ and Rad52 are recruited to repair the double-strand break. The 2-nt, 3' DNA overhang generated by the I-TevI nuclease domain of TevSaCas9 hybridizes with the 3' end of a co-localized repair template guide RNA to specifically license repair. Substitutions that destabilize the repair duplex reduce repair efficiency. Targeted RNA-templated repair (rep-editing) harnesses cellular RNA-based DNA repair pathways to introduce precise nucleotide edits, deletions and insertions in human cells with high efficiency and fidelity independent of co-delivered repair functions. The small size of TevSaCas9 and RNA repair template offers delivery advantages over size-constrained or multi-component editing systems.

show abstract

Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks

Cited by 16 publications

References 83 publications

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington’s disease

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington’s disease

Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways

Harnessing RNA-based DNA repair pathways for targeted gene editing

Contact Info

Product

Resources

About