The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by prominent cellular infiltrates in the bowel wall. Chemokines are chemotactic cytokines that are able to promote leukocyte migration to areas of inflammation and are also able to initiate cell activation events. They have recently been implicated in the pathophysiology of many disease states. The aim of this study was to detail the degree and distribution of specific chemokines, interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, -2, and -3, and macrophage inflammatory protein (MIP)-1alpha and -1beta, in IBD mucosa. Thirty-nine patients were included, ten controls, 20 ulcerative colitis (UC), and nine Crohn's disease (CD), with a range of disease activity. Colonic mucosal biopsies were collected from UC, CD, and control patients and embedded in glycol methacrylate. Two-micrometre-thick sections were cut and stained using immunohistochemistry for chemokine protein expression. Sections were analysed using a light microscope. Expression of all types of chemokine protein was detected in colonic mucosa from both control and IBD patients. Patterns of staining between IBD patients and controls differed significantly, but CD and UC patients demonstrated similar patterns of staining. Individual chemokine expression was found to be significantly up-regulated in IBD when patients were compared with the non-diseased group in all areas of the mucosal sections. Up-regulated chemokine expression correlated with increasing activity of the disease. It is concluded that human colonic chemokine expression is non-selectively up-regulated in IBD. The results supported the hypothesis that the degree of local inflammation and tissue damage in UC and CD is dependent on local expression of specific chemokines within IBD tissues.

The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3(-/-) chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3(-/-) cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.

1 . This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.DNA strand breakage can result in chromosomal rearrangement and is a major threat to genetic stability. Of particular threat are breaks that arise from damaged DNA bases, several thousand of which occur spontaneously per cell each day (20). The most common such breaks are single-strand breaks, which are formed as intermediates of base excision repair. The threat to genetic stability from DNA strand breaks that arise from base damage is illustrated by the phenotype of rodent cells that harbor mutations within the DNA repair gene XRCC1. XRCC1 is essential for embryonic development in mice (35), and XRCC1 mutant mouse or CHO cells that possess little or no XRCC1 protein exhibit increased frequencies of spontaneous sister chromatid exchange and chromosomal aberration (6,7,12,29,35,39). XRCC1 mutant cells appear unable to efficiently rejoin DNA single-strand breaks resulting from either endogenous base damage (35) or that induced by ionizing radiation or alkylating agents (36,37,39). Sequence analysis has not provided any indication of the role of XRCC1 in single-strand break repair (SSBR). However, biochemical studies have revealed that this protein interacts with DNA ligase III and DNA polymerase ␤ (6-8, 18). Thus, it is possible that XRCC1 functions as a molecular chaperone or scaffold protein, stabilizing and/or modifying the activity of other polypeptides. For example, the interaction of XRCC1 with DNA ligase III appears to be required for normal cellular levels of the latter, and reduced levels of DNA ligase III can result in inefficient SSBR during the excision repair of abasic sites in vitro (6,7,11). This interaction is mediated by a C-terminal BRCT domain in XRCC1, designated BRCT II (23, 34). BRCT domains have been identified in more than 40 polypeptides, defining a novel protein superfamily, and typically span 80 to 100 amino acids (5, 10). The function of these structures appears to involve, but may not be restricted to, the mediation of protein-protein interactions (5,10,23,34). In the present study, we have examined the importance of the XRCC1 BRCT II domain and its interaction with DNA ligase III to SSBR in Chinese hamster ovary (CHO) cells. MATERIALS AND METHODS Expression constructs, cell lines, and cell synchrony. A mutant XRCC1pmBRCT open reading frame (ORF) was generated by subcloning constructs described previously (34) and was inserted into the mammalian expression vector pcD2E (17). All XRCC1 ORFs encode a decahistidine tag at the C terminus. XRCC1 mutant EM9 cells were transfected with pcD2E or pcD2E harboring XRCC1 by electroporation, and Ͼ50 independent G418 r clones were pooled to generate the cell lines EM9-X, EM9-X pmBRCT , and EM9-V. Synchrony in G 1 was achieved via serum starvation and incubation in mimosine (25). Cells were incubated sequentially in ␣-MEM plus 10% fetal bovine serum (FBS) for 12 to 15 h, ␣-MEM plus 0.1% FBS fo...