Several single-base substitution mutations have been introduced into the lacZa gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a sitespecific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, %10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung')

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DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5 CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI؊ tumors, as well as in MSI؉ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

▪ Abstract  DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

Letter to the Editor Letter to the Editor amino acid sequence identity and similarity amongst them-The Y-Family of DNA Polymerases UmuC/DinB/Rev1/Rad30 DNA polymerases be referred ESBS Boulevard S. Brant to as the "Y-family" of DNA polymerases.Phylogenetic analysis of the Y-family of DNA poly-67400 Strasbourg France merases reveals several branches to an unrooted tree (Figure 1). Interestingly, not all branches are evenly dis-

Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine if ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active site methionine in yeast DNA polymerase ε (Pol ε). Compared to wild type Pol ε, ribonucleotide incorporation in vitro was 3-fold lower for M644L and 11-fold higher for M644G Pol ε. This hierarchy was re-capitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S-phase, had elevated dNTP pools and generated 2–5 base pair deletions in repetitive sequences at a high rate and gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair, and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36-to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols α, δ, and ε, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...