‘Saccharomyces cerevisiae MSH2/6 Complex Interacts with Holliday Junctions and Facilitates Their Cleavage by Phage Resolution Enzymes

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies

Modulation of the DNA-binding activity of Saccharomyces cerevisiae MSH2–MSH6 complex by the high-mobility group protein NHP6A, in vitro

DNA mismatch repair corrects mispaired bases and small insertions/deletions in DNA. In eukaryotes, the mismatch repair complex MSH2–MSH6 binds to mispairs with only slightly higher affinity than to fully paired DNA in vitro. Recently, the high-mobility group box1 protein, (HMGB1), has been shown to stimulate the mismatch repair reaction in vitro. In yeast, the closest homologs of HMGB1 are NHP6A and NHP6B. These proteins have been shown to be required for genome stability maintenance and mutagenesis control. In this work, we show that MSH2–MSH6 and NHP6A modulate their binding to DNA in vitro. Binding of the yeast MSH2–MSH6 to homoduplex regions of DNA significantly stimulates the loading of NHP6A. Upon binding of NHP6A to DNA, MSH2–MSH6 is excluded from binding unless a mismatch is present. A DNA binding-impaired MSH2–MSH6F337A significantly reduced the loading of NHP6A to DNA, suggesting that MSH2–MSH6 binding is a requisite for NHP6A loading. MSH2–MSH6 and NHP6A form a stable complex, which is responsive to ATP on mismatched substrates. These results suggest that MSH2–MSH6 binding to homoduplex regions of DNA recruits NHP6A, which then prevents further binding of MSH2–MSH6 to these sites unless a mismatch is present

Characterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87119/1/j.1399-0004.2010.01594.x.pd

A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed

Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways

Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA)

Mismatch Repair (MMR) is closely linked to DNA replication; however, other than the role of the replicative sliding clamp (PCNA) in various MMR functions, the linkage between DNA replication and MMR has been difficult to investigate. Here we use an in vitro DNA replication system based on simian virus 40, to investigate MMR recruitment to replicating DNA. Both DNA replication and MMR proteins are recruited to replicating DNA in an origin-dependent fashion. Primer synthesis is required for recruitment of both PCNA and MMR proteins, but not for recruitment of the single-stranded DNA-binding protein (RPA). Blocking PCNA recruitment to replicating DNA with a p21-based polypeptide blocks PCNA and MMR, but not RPA recruitment. Once PCNA and subsequent proteins required for replication are loaded onto DNA, addition of p21 leaves PCNA on the replicating DNA, but actively displaces MMR proteins. These findings indicate that the MMR machinery is recruited to replicating DNA through its interaction with PCNA, and suggests that this occurs via binding of the MMR proteins to the multi-protein interaction sites on PCNA. These studies demonstrate the utility of this system for further investigation of the role of DNA replication in MMR

Mutation Rates of TGFBR2 and ACVR2 Coding Microsatellites in Human Cells with Defective DNA Mismatch Repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFβ family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1−/−, hMSH6−/−, hMSH3−/−, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a −1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7–35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a −1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91×10−4) and 15 (2.18×10−4) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was ∼3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The −1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks