Base excision repair is the system used from bacteria to man to remove the tens of thousands of endogenous DNA damages produced daily in each human cell. Base excision repair is required for normal mammalian development and defects have been associated with neurological disorders and cancer. In this paper we provide an overview of short patch base excision repair in humans and summarize current knowledge of defects in base excision repair in mouse models and functional studies on short patch base excision repair germ line polymorphisms and their relationship to cancer. The biallelic germ line mutations that result in MUTYH-associated colon cancer are also discussed.

Cells transduce mechanical forces into biochemical signals; traditionally these processes are thought to occur through direct effects on the cell membrane, the cytoskeleton, or specific transmembrane proteins. In multicellular tissues mechanical forces alter intercellular spacing through redistribution of interstitial fluid. Recent morphological and biochemical observations, bolstered by analytical modeling, support a new paradigm for mechanotransduction arising from constitutive growth factor shedding into a dynamically regulated interstitial volume.

The X Family of DNA polymerases in eukaryotic cells consists of Terminal Transferase, and DNA polymerases β, λ, and μ. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases β, λ, and μ. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.

14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol ␤ variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.

Thirty percent of the 189 tumors studied to date express DNA polymerase ␤ variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein.Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorageindependent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase ␤ variant proteins, implying that it has a mutational basis. Because DNA polymerase ␤ functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.base excision repair ͉ DNA repair ͉ mutagenesis S pontaneous DNA damage occurs at a rate of Ϸ10,000 lesions per cell per day, and much of this damage is repaired by the base excision repair (BER) machinery (1, 2). The BER system plays a critical role in maintaining cellular genomic stability. During BER, damaged bases are removed by a DNA glycosylase, followed by incision of the DNA by AP endonuclease (APE) at a position that is usually 5Ј to the lesion, leaving a nick with a 3ЈOH and a 5Ј deoxyribose phosphate (2). DNA polymerase beta (pol ␤) binds to the nick, removes the deoxyribose phosphate with its DRP lyase activity (3), and fills in the single nucleotide gap, using its DNA polymerase activity (4).Fifty-eight of the 189 tumors characterized to date express DNA pol ␤ variant proteins (for review, see ref. 5) (6). Of these, 28 (48%) expressed variants with single amino acid alterations, seven expressed truncated forms of pol ␤, and eight expressed multiple variant forms of pol ␤. These mutations are absent from normal tissue from the same individuals and are not among the common polymorphisms found within the pol ␤ gene (http:͞͞ egp.gs.washington.edu͞data͞polb) (5, 7). In addition, an alternative splice variant of pol ␤ in which exon 11 is deleted was expressed in 15 tumors. This splice variant appears to interfere with BER (8). This exon 11 splice variant has been detected in normal tissue, including normal tissue isolated from 2 of 15 patients with tumors, and its link to cancer etiology remains controversial (9-12). Each of the tumors characterized to date also contain the wild-type (WT) pol ␤ allele.The I260M variant of pol ␤ was identified in a prostate carcinoma (13). Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymeras...

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity. SOS mutagenesis is due to an error-prone mode of DNA replication expressed in wild-type Escherichia coli after exposure to UV light or to chemical agents that block DNA replication and induce the SOS response. In certain mutant strains in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity by which spontaneous mutation rates are elevated as much as 50-fold. In either case, SOS mutagenesis depends on the products of the recA, lexA, and umuDC genes. RecA and LexA proteins regulate the SOS response, and UmuDC proteins are believed to be essential for the actual mutagenic event (for reviews, see references 36 and 37).RecA protein has two essential roles in SOS mutagenesis. One is its regulatory role. DNA damage generates a signal that results in an altered form of RecA protein (RecA*). RecA* causes the proteolytic cleavage of LexA, the repressor of some 20 genes composing the SOS regulon, including recA itself and the umuDC operon (for reviews, see references 19 and 28). Amplification of UmuDC is necessary but not sufficient for SOS mutagenesis, as shown by the absence or weak expression of SOS mutator activity in recA+ strains carrying a lexA(Def) mutation that inactivates LexA. Such strains become strong mutators only if the recA+ allele is replaced by recA441 or recA730, which encode spontaneously activated RecA proteins, indicating that RecA* has an essential role in SOS mutagenesis other than its antirepressor function (la, 11, 38). It has recently been shown that RecA* promotes the proteolytic cleavage of UmuD protein both in vitro (5) and in vivo (32) and that only the larger COOH-terminal fragment (UmuD') is active in UV mutagenesis, whereas the unprocessed UmuD protein is not (27). SOS

Previous investigations have shown that Ϸ35% of the 90 tumors analyzed to date contain mutations within the DNA polymerase ␤ (pol ␤) gene. The existence of pol ␤ mutations in a substantial fraction of human tumors studied suggests a link between DNA pol ␤ and cancer. A DNA pol ␤ variant, in which Lys-289 has been altered to Met, was identified previously in a colorectal carcinoma. The K289M protein was expressed in mouse L cells containing the cII mutational target. The DNA was packaged and used to infect bacterial cells to obtain the spontaneous mutation frequency. We found that expression of K289M in the mouse cells resulted in a 2.5-fold increase in the mutation frequency. What was most interesting was that expression of K289M in these cells resulted in a 16-fold increase in the frequency of C to G or G to C base substitutions at a specific site within the cII target. By using this cII target sequence, kinetic analysis of the purified K289M protein revealed that it was able to misincorporate dCTP opposite template C and dGTP opposite template G with significantly higher efficiency than the wild-type pol ␤ protein. We provide evidence that misincorporation of nucleotides by K289M results from altered positioning of the DNA within the active site of the enzyme. Our data are consistent with the interpretation that misincorporation of nucleotides resulting from altered DNA positioning by the K289M protein has the potential to result in tumorigenesis or neoplastic progression.M utations in the gene encoding DNA polymerase ␤ (pol ␤) have been identified in human colorectal, prostate, lung, and breast carcinomas and mouse lymphomas (1-5). Thus far, only 90 tumors have been analyzed for mutations within the pol ␤ coding sequence, and mutations are present in 35% of these tumors. The pol ␤ tumor-associated mutations are found only in the tumor, and not in normal tissue from the same patient, implying that they represent sporadic mutations underlying neoplastic disease. Furthermore, the mutations identified in these tumors are not present in the pol ␤ gene of 124 normal individuals (6). Also of interest is that pol ␤ is located within the proximal region of the short arm of chromosome 8 (p12-p11), a region that is frequently lost in a variety of human tumors, including colorectal and prostate carcinomas (7). These studies suggest a link between mutations within the pol ␤ gene and carcinogenesis. Another piece of evidence that is consistent with a role for pol ␤ in cancer is its interaction with the tumor suppressor protein p53 (8, 9). The p53 protein stabilizes pol ␤ at an abasic site. An alteration of the p53-pol ␤ interaction could result in less efficient DNA repair, which may contribute to the development of neoplastic disease. Most interestingly, a pol ␤ mutant with an 87-bp deletion, which has been found in primary colorectal, lung, and breast adenocarcinomas (3,5,10), is dominant to the WT enzyme and disrupts its base excision repair (BER) activity if expressed in human cell lines (11,12). It is quite possible t...

Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase ␤ (Pol ␤), which catalyze the first three steps in BER, are able to process their substrates in both 601-and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase III␣-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase III␣-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol ␤ on nucleosome substrates. Collectively, these findings provide insights into ratelimiting steps that govern BER in chromatin and reveal a unique role for ligase III␣-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.Reactive oxygen species (ROS), generated as by-products of normal aerobic cellular metabolism or from exposure to exogenous agents, such as gamma irradiation, generate approximately 20,000 DNA damage events per day in each nucleated human cell. The DNA lesions produced include numerous oxidative base damages, apurinic/apyrimidinic (AP) sites, and single-strand DNA breaks (6). Base excision repair (BER) enzymes recognize and replace oxidized bases with the corresponding undamaged bases. In its simplest ("short-patch") form, BER entails four enzymatic steps (1,10,21,23,51,53) (Fig. 1A), beginning with the recognition and excision of a damaged base by either a mono-or bifunctional DNA glycosylase. Bifunctional glycosylases first cleave the glycosidic bond between the damaged base and the deoxyribose and then cleave the phosphodiester bond 3Ј of the resulting AP site. AP endonuclease (APE) removes a residual moiety to generate a single nucleotide gap, with a 3Ј-OH group that can be filled by DNA polymerase ␤ (Pol ␤). Finally, DNA ligase III-␣ (LigIII␣), in association with XRCC1, catalyzes the formation of a phosphodiester bond between the 3Ј-OH of the newly added nucleotide and the adjacent downstream 5Ј-phosphate.The nucleosomes that package most of the nuclear DNA in eukaryotes provide only minimal protection from ROS (14, 31); a small degree of protection from hydroxyl radicals is evident in DNA segments where the minor groove faces into the histone octamer (20), and histones themselves may act as a sink for ROS, thereby reducing the frequency of free-radicalinflicted DNA damage (28). Clearly, however, nucleosomal DNA is vulnerable to oxidative damage that must be made available to BER enzymes. Chromatin remodeling agents and histone chaperones facilitate most processes involving chromatin, and the other DNA repair pathways-nucleotide excision repair, mismatc...