MSH2-dependent Mismatch Repair Research Articles

FEN1-mediated α-segment error editing during Okazaki fragment maturation

DNA-replication-repair machinery determines the non-random occurrence of variations across the genome. During eukaryotic nuclear-genome replication, most parts of the leading and lagging strand are synthesized by high fidelity DNA polymerase ϵ and δ respectively. However, a third enzyme polymerase (Pol) α-primase initiates each synthesis event. During Okazaki fragment, down-stream of the RNA primer and synthesis, DNA fragment synthesized by proofreading-deficient Pol-α, which we termed as α-segment, provide major source of mutations and undergo DNA repair process to remove the incorporated errors.1 The α-segment contributes ∼1.5% of total genome. A series of the very recent papers made available by Nature press indicates that despite Okazaki fragment processing, DNA synthesized by Pol α is retained in vivo.2-5 DNA-binding proteins including histones and transcription factors that rapidly re-associate, post-replication, to act as partial barriers to Pol-δ-mediated displacement of the α-segment, resulting increased mutation rates in the region. What is the cellular mechanism that minimizes the mutation frequency in the alpha-segment? Limited evidence is available. Pol α variant strains have mild mutator phenotypes, resulting from DNA replication errors generated by Pol α, and their mutation rates are synergistically increased by loss of MSH2-dependent mismatch repair (MMR). Thus, MSH2-dependent MMR in yeast plays a major role in correcting mismatches in the α-segment.1 Additional study indicated that some mismatches generated by yeast Pol α are excised by exonuclease 1 (EXO1), a 5′ exonuclease involved in MMR.6 Interestingly, the loss of MMR resulting from deleting EXO1 is mild compared to the loss of MMR from deleting MSH2, implying the existence of a MSH2-dependent but EXO1-independent mechanism that removes mismatches from the α-segment. In our paper currently in press with EMBO Journal,7 we investigated the molecular mechanism mediated by FEN1 and MSH2 functional complex to edit the errors in the α-segment. Our unique substrate designing strategy segregates the RNA primer removal (RPR) and α-segment error editing (AEE) activities of FEN1 and characterize efficiency of functional enzymatic component of nuclear extract (NEs). Interestingly, besides its well-characterized RNA primer removl function, FEN1 was able to go on in an exonuclease fashion in presence of the mis-paired nucleotides in the α-segment and remove them within a limited distance (12 nts) from the nick MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 AEE defective mutations display a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility.7 The proposed AEE mechanism is significant because it effectively removes replication errors while concomitantly maturing Okazaki fragments, which is a rate-limiting step in DNA replication.7 However, if a mutation occurs in AEE components that deregulates the process and leads to constitutive editing and futile cycles, it could affect DNA ligation and result in spontaneous DNA strand breaks and consequently, various forms of chromosome aberrations. Such a mutation would be different from the ones due to defects in RPR process, of which the major form is the duplication mutation. Both the mutations that result in DNA strand breaks and aneuploidy and the duplication mutation, are hallmarks of human cancer. The current study identifies a novel role of FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Activation Induced Cytidine Deaminase-Associated DNA Repair Pathways Influence Germinal Center B Cell Lymphomagenesis

Somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation induced cytidine deaminase (AID). Resulting uracil-guanine (U-G) mismatches are processed by UNG-dependent base-excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with B cell lymphomas, the role of downstream AID-associated DNA repair pathways in lymphomagenesis is not defined. Through deregulated expression of BCL6, IµHABcl6 mice develop an AID-dependent GC-derived lymphoma that resembles human diffuse large B cell lymphoma (DLBCL). We have previously demonstrated that IµHABcl6 Ung-/-Msh2-/- mice have a similar incidence (35% vs 27%) but a 2.5-fold shorter median time to development of B220+ IgM+ PNAhi CD138- DLBCL compared with IµHABcl6 mice (6.5 months vs. 16.2 months; P = 0.0003). This suggests that AID-associated DNA repair pathways serve to protect the GC B cell and delay BCL6-driven lymphomagenesis. To investigate the individual contribution of BER and MMR in the pathogenesis of GC-derived lymphoma, we have now generated IµHABcl6 Ung-/- and IµHABcl6 Msh2-/- single-deficient mice. The majority of IµHABcl6 Ung-/- mice remained healthy beyond 20 months with only 3 of 22 (13.6%) mice becoming sick starting at ∼16 months. Sick mice were found to have splenic lymphomas comprised of mature B220+ IgM+ PNAlow CD138- B cells. Histological examination revealed expanded follicles with a population of small lymphocytes, consistent with a follicular B cell lymphoma which has been shown to arise in Ung-/- mice. In contrast, 18 of 22 (81.8%) IµHABcl6 Msh2-/- mice rapidly succumbed to malignancy starting at ∼3 months and had a median survival of 6 months. Of 15 tumors available for analysis, there was 1 histiocytic sarcoma, 1 squamous cell carcinoma, 4 T cell lymphomas, and 9 B220+ IgM- PNA- CD138- pre-B cell lymphomas (determined by histology, immunophenotyping and gene expression profiling). None of the IµHABcl6 Ung-/- or IµHABcl6 Msh2-/- mice developed DLBCL. Since lack of UNG is strongly protective when MSH2 is present, we conclude that in the setting of deregulated BCL6, UNG promotes the development of DLBCL. In contrast, MSH2 is protective against the development of tumors in general and does not facilitate DLBCL in the absence of UNG. Combined with the observation that IµHABcl6 Ung-/-Msh2-/- mice develop DLBCL with a significantly shorter latency than IµHABcl6 mice, this data indicates that a complex interplay between AID-associated BER and MMR produces a net protective effect against lymphomagenesis. In the absence of UNG and MSH2, AID-generated U-G mismatches are not processed into strand lesions and are simply replicated, yielding C/G to T/A transition mutations. Thus, to assess how combined lack of UNG and MSH2 might promote the accelerated development of BCL6-driven lymphoma, we carried out spectral karyotyping and sequence analysis of AID target genes (IgJH4, cMyc, Pim1, RhoH, Cd79a, CD79b, H2afx, Pax5, and Cd83) in lymphomas from the different genotypes. IµHABcl6 DLBCLs (3/3) harbored various complex chromosome abnormalities, consistent with previous findings. Numerous clonal and sub-clonal chromosome abnormalities including translocations, duplications, deletions, and aneuploidies were also detected in IµHABcl6 Ung-/-Msh2-/- (4/4) and IµHABcl6 Ung-/- (2/2) lymphomas. Pre-B cell tumors from IµHABcl6 Msh2-/- mice could not be stimulated to produce metaphase chromosomes. Clonal and non-clonal mutations of the IgJH4 intronic region were identified in lymphomas from IµHABcl6 (2/3), IµHABcl6 Ung-/-Msh2-/- (4/4), and IµHABcl6 Ung-/- (2/3) mice, consistent with ongoing AID activity. No mutations were detected in 3 pre-B cell lymphomas, consistent with their pre-GC origin. Six clonal mutations within AID hotspots (all C/G to T/A) were identified in Pim1, RhoH, and Pax5 in 2 of 4 IµHABcl6 Ung-/-Msh2-/- DLBCLs. None of the other genotypes carried any clonal mutations of non-Ig genes. Thus, chromosome abnormalities in GC B cell lymphomas can arise through mechanisms independent of BER and MMR but may be due to off-target effects of AID on other genes that regulate cell cycle, apoptosis, or genomic stability. Disclosures:No relevant conflicts of interest to declare.

The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Abstract 397Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER (Ung−/−) and MMR (Msh2−/−). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung−/−Msh2−/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung−/−Msh2−/−mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p<0.0001). All 5 of the Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung−/−Msh2−/−mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung−/−Msh2−/−mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung−/−Msh2−/−tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung−/−Msh2−/−mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung−/−Msh2−/−tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes (cMyc, Pim1, RhoH, Pax5, Cd79a, Fas, H2ax and OcaB) in tumors from 3 Iμ-Bcl6 Ung−/−Msh2−/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10−4, suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung−/− and Iμ-Bcl6 Msh2−/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures:No relevant conflicts of interest to declare.

Msh2-dependent DNA repair mitigates a unique susceptibility of B cell progenitors to c-Myc -induced lymphomas

C-Myc is one of the most common targets of genetic alterations in human cancers. Although overexpression of c-Myc in the B cell compartment predisposes to lymphomas, secondary mutations are required for disease manifestation. In this article, we show that genetic deficiencies causing arrested B cell development and accumulation of B cell progenitors lead to accelerated lymphomagenesis in Emu c-myc transgenic mice. This result suggests that B cell progenitors are more prone than their mature counterparts to developing secondary oncogenic lesions that complement c-Myc in promoting transformation. To investigate the nature of these oncogenic lesions, we examined Emu c-myc mice deficient in mismatch repair function. We report that Msh2(-/-) Emu c-myc and Msh2(G674A/G674A) Emu c-myc mice rapidly succumb to pro-B cell stage lymphomas, indicating that Msh2-dependent mismatch repair function actively suppresses c-Myc-associated oncogenesis during early B cell development.

Characterization of palindromic loop mismatch repair tracts in mammalian cells

Single- and multi-base (loop) mismatches can arise in DNA by replication errors, during recombination, and by chemical modification of DNA. Single-base and loop mismatches of several nucleotides are efficiently repaired in mammalian cells by a nick-directed, MSH2-dependent mechanism. Larger loop mismatches (≥12 bases) are repaired by an MSH2-independent mechanism. Prior studies have shown that 12- and 14-base palindromic loops are repaired with bias toward loop retention, and that repair bias is eliminated when five single-base mismatches flank the loop mismatch. Here we show that one single-base mismatch near a 12-base palindromic loop is sufficient to eliminate loop repair bias in wild-type, but not MSH2-defective mammalian cells. We also show that palindromic loop and single-base mismatches separated by 12 bases are repaired independently at least 10% of the time in wild-type cells, and at least 30% of the time in MSH2-defective cells. Palindromic loop and single-base mismatches separated by two bases were never repaired independently. These and other data indicate that loop repair tracts are variable in length. All tracts extend at least 2 bases, some extend <12 bases, and others >12 bases, on one side of the loop. These properties distinguish palindromic loop mismatch repair from the three known excision repair pathways: base excision repair which has one to six base tracts, nucleotide excision repair which has ∼30 base tracts, and MSH2-dependent mismatch repair, which has tracts that extend for several hundred bases.

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1 −/− mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1 −/− mice. Interestingly, the net frequency of rpsL − forward mutants showed no apparent increase in MTH1 −/− mice as compared to MTH1 +/+ mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL − mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T→C:G transversion than the wild type cells, an increase in frequency of A:T→C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1 −/− mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1 −/− mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C→T:A transversions was observed with MTH1 −/− MSH2 −/− mice over MSH2 −/− mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.

Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.

A Novel Mutation Avoidance Mechanism Dependent on S. cerevisiae RAD27 Is Distinct from DNA Mismatch Repair

Mutations in the S. cerevisiae RAD27 (also called RTH1 or YKL510) gene result in a strong mutator phenotype. In this study we show that the majority of the resulting mutations have a structure in which sequences ranging from 5–108 bp flanked by direct repeats of 3–12 bp are duplicated. Such mutations have not been previously detected at high frequency in the mutation spectra of mutator strains. Epistasis analysis indicates that RAD27 does not play a major role in MSH2-dependent mismatch repair. Mutations in RAD27 cause increased rates of mitotic crossing over and are lethal in combination with mutations in RAD51 and RAD52. These observations suggest that the majority of replication errors that accumulate in rad27 strains are processed by double-strand break repair, while a smaller percentage are processed by a mutagenic repair pathway. The duplication mutations seen in rad27 mutants occur both in human tumors and as germline mutations in inherited human diseases.

Requirement of the Yeast MSH3 and MSH6 Genes for MSH2-dependent Genomic Stability

Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.

Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.