Domain analysis of PNKP–XRCC1 interactions: Influence of genetic variants of XRCC1

Abstract

Edited by Patrick SungPolynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the singlestrand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166 -436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.

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

“…1C ) , suggesting that their interaction is direct, and is mediated through the FHA domain of PNKP. This is consistent with previous reports of interaction of DNA repair proteins, XRCC1 33 and XRCC4 17 with PNKP through its FHA domain.…”

“…Nuclear extracts were prepared following the protocol used for Co-IP studies. For kinase activity assay, γP 32 labeled ATP was incubated in kinase assay buffer (80 mM succinic acid pH 5.5, 10 mM MgCl2, 1 mM DTT, 2.5% glycerol) along with 1.0 μg/μl acetylated BSA, and 0.6 pmol labeled substrate for 30 min at 30 o C 34 . 100 fmol of PNKP and 2.5 pmole of cold substrate were used in this assay.…”

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

“…K142 acetylation (AcK142) was found to be constitutive and identified in both control and Bleo-treated cells, whereas K226 acetylation (AcK226) was induced only upon Bleo treatment. These two distinct acetylation sites were found to be located in different domains of PNKP ( 9 , 35 , 38 , 39 ): AcK142 in the linker region (111–144 aa), and AcK226 in the phosphatase domain (145–336 aa) ( Supplementary Figure S1D ). To validate the acetylation sites in PNKP and the differential mode of their acetylation, we further performed the MS analysis of affinity-purified FLAG-tagged PNKP from WT and K226R (acetylation-deficient mutant) PNKP expressing HEK293 cells following mock and Bleo-treatment.…”

CDKs-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki Fragments and genome stability

“…It has 3′ -phosphatase and 5′ -kinase activity (Chappell et al, 2002; Jilani et al, 1999; Karimi-Busheri et al, 1999). PNKP is recruited to the sites of SSBs and DSBs depending on the interactions with XRCC1 and XRCC4, respectively, and is involved in base excision repair (BER), SSB repair, and non-homologous end joining (NHEJ) for DSB repair (Breslin and Caldecott, 2009; Mani et al, 2019; Mani et al, 2010; Tsukada et al, 2020a; Tsukada et al, 2020b). Human PNKP consists of 521 amino acids, including the forkhead-associated (FHA) domain (amino acid residue 1–110) in the amino-terminal region and phosphatase (146–337) and kinase (341–516) domains in the carboxy-terminal region, which are connected by a linker region (111–145).…”

“…Human PNKP consists of 521 amino acids, including the forkhead-associated (FHA) domain (amino acid residues 1–110) in the amino-terminal region, and phosphatase (146–337) and kinase (341–516) domains in the carboxy-terminal region, which are connected by a linker region (111–145). PNKP is recruited to SSBs and DSBs sites depending on its interactions via the FHA domain of PNKP with XRCC1 and XRCC4, respectively, and is involved in base excision repair (BER), SSB repair, and non-homologous end joining (NHEJ) for DSB repair 4, 5, 6, 7, 8 . The PNKP linker region includes a nuclear localization signal and phosphorylation sites 7 .…”