p38 MAPK signaling and phosphorylations in the BRCT1 domain regulate XRCC1 recruitment to sites of DNA damage.

XRCC1 is a scaffold protein involved in base excision repair and single strand break repair. It is a phosphoprotein that contains more than 45 phosphorylation sites, however only a few of these have been characterized and connected to specific kinases and functions. Mitogen activated protein kinases (MAPK) are mediators of cellular stress responses, and here we demonstrate that p38 MAPK signaling is involved in phosphorylation of XRCC1 and regulation of recruitment to oxidative stress. Inhibition of p38 MAPK caused a marked pI shift of XRCC1 towards a less phosphorylated state. Inhibition of p38 also increased the immediate accumulation of XRCC1 at site of DNA damage in a poly(ADP)-ribose (PAR) dependent manner. These results suggest a link between PARylation, p38 signaling and XRCC1 recruitment to DNA damage. Additionally, we characterized two phosphorylation sites, T358 and T367, located within, or close to, the phosphate-binding pocket of XRCC1, which is important for interaction with PAR. Mutation of these sites impairs recruitment of XRCC1 to DNA damage and binding to PARP1/PAR. Collectively, our data suggest that phosphorylation of T358 and T367 and p38 signaling are important for proper regulation of XRCC1 recruitment to DNA damage and thereby avoidance of potential toxic and mutagenic BER-intermediates.

Acute exhaustive aerobic exercise training impair cardiomyocyte function and calcium handling in Sprague-Dawley rats.

INTRODUCTION: Recent data from long-distance endurance participants suggest that cardiac function is impaired after completion. Existing data further indicate that right ventricular function is more affected than left ventricular function. The cellular mechanisms underpinning cardiac deterioration are limited and therefore the aim of this study was to examine cardiomyocyte and molecular responses of the right and left ventricle to an acute bout of exhaustive endurance exercise. MATERIALS AND METHODS: Male Sprague-Dawley rats were assigned to sedentary controls or acute exhaustive endurance exercise consisting of a 120 minutes long forced treadmill run. The contractile function and Ca2+ handling properties in isolated cardiomyocytes, protein expression levels of sarcoplasmic reticulum Ca2+-ATPase and phospholamban including two of its phosphorylated states (serine 16 and threonine 17), and the mitochondrial respiration in permeabilized cardiac muscle fibers were analyzed. RESULTS: The exercise group showed a significant reduction in cardiomyocyte fractional shortening (right ventricle 1 Hz and 3 Hz p<0.001; left ventricle 1 Hz p<0.05), intracellular Ca2+ amplitude (right ventricle 1 and 3 Hz p<0.001; left ventricle 1 Hz p<0.01 and 3 Hz p<0.05) and rate of diastolic Ca2+ decay (right ventricle 1 Hz p<0.001 and 3 Hz p<0.01; left ventricle 1 and 3 Hz p<0.01). Cardiomyocyte relaxation during diastole was only significantly prolonged at 3 Hz in the right ventricle (p<0.05) compared to sedentary controls. We found an increase in phosphorylation of phospholamban at serine 16 and threonine 17 in the left (p<0.05), but not the right, ventricle from exhaustively exercised animals. The protein expression levels of sarcoplasmic reticulum Ca2+-ATPase and phospholamban was not changed. Furthermore, we found a reduction in maximal oxidative phosphorylation and electron transport system capacities of mitochondrial respiration in the right (p<0.01 and p<0.05, respectively), but not the left ventricle from rats subjected to acute exhaustive treadmill exercise. CONCLUSION: Acute exhaustive treadmill exercise is associated with impairment of cardiomyocyte Ca2+ handling and mitochondrial respiration that causes depression in both contraction and diastolic relaxation of cardiomyocytes.

X-ray repair cross complementing protein 1 in base excision repair.

X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1.

XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166-436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLß or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein.

XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage.

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil.