Liya Gu

Identification and characterization of OGG1 mutations in patients with Alzheimer's disease

Patients with Alzheimers disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C796) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C796-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease.

Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability.

Breast cancer is the most common cancer in women, but its pathogenesis is still unclear. Microsatellite instability (MSI) has been identified in breast cancer cells, suggesting an association with mismatch repair defects. To test this hypothesis, we investigated MSI, protein expression of hMSH2 and hMLH1, as well as genetic and epigenetic modifications of these two genes in 32 sporadic breast tumors. MSI was identified in 15 cases. Immunohistochemistry analysis revealed that all MSI cases but one had lower than normal expression of hMSH2 (nine cases), hMLH1 (12 cases), or both (seven cases). In tumors with MSI, both genetic and epigenetic modifications of these mismatch repair genes were also identified. Eight cases harbored mutations or polymorphisms in hMSH2 and hMLH1, and 10 exhibited hypermethylation in the promoter region of hMLH1. These results suggest that both genetic and epigenetic alterations of hMSH2 and especially of hMLH1 contribute to genomic instability and tumorigenesis in sporadic breast cancer.

Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A

ABSTRACT DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.

hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono‐ and dinucleotide sequences. Furthermore, RNA‐interference‐mediated hMRE11‐knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC‐associated hMLH1 missense mutations located within the hMRE11‐interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near‐complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1–hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.

Evidence That Nucleosomes Inhibit Mismatch Repair in Eukaryotic Cells

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSα and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSα specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSα displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSα sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.

Bi-directional Processing of DNA Loops by Mismatch Repair-dependent and -independent Pathways in Human Cells

Previous work has shown that small DNA loop heterologies are repaired not only through the mismatch repair (MMR) pathway but also via an MMR-independent pathway in human cells. However, how DNA loop repair is partitioned between these pathways and how the MMR-independent repair is processed are not clear. Using a novel construct that completely and specifically inhibits MMR in HeLa extracts, we demonstrate here that although MMR is capable of bi-directionally processing DNA loops of 2, 4, 5, 8, 10, or 12 nucleotides in length, the repair activity decreases with the increase of the loop size. Evidence is presented that the largest loop that the MMR system can process is 16 nucleotides. We also show that strand-specific MMR-independent loop repair occurs for all looped substrates tested and rigorously demonstrate that this repair is bi-directional. Analysis of repair intermediates generated by the MMR-independent pathway revealed that although the processing of looped substrates with a strand break 5′ to the heterology occurred similarly to MMR (i.e. excision is conducted by exonucleases from the pre-existing strand break to the heterology), the processing of the heterology in substrates with a 3′ strand break is consistent with the involvement of endonucleases.

Regulation of replication protein A functions in DNA mismatch repair by phosphorylation.

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase δ-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.

Effect of Carcinogenic Acrolein on DNA Repair and Mutagenic Susceptibility

Background: Acrolein is highly reactive and abundant in tobacco smoke. Results: Acrolein induces DNA damage, inhibits excision repair and mismatch repair, causes repair protein degradation, and enhances mutagenesis. Conclusion: Acrolein induces DNA damage and inhibits DNA repair that causes mutagenesis and initiates carcinogenesis. Significance: This is the first demonstration that acrolein inhibits DNA repair pathways by induction of repair protein degradation. Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N2-cyclic propano-2′-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.

Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts.

Expansion of CAG/CTG trinucleotide repeats is associated with certain familial neurological disorders, including Huntingtons disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism contributes to their expansion. However, the molecular mechanism(s) by which cells remove CAG/CTG hairpins remain unknown. Here we demonstrate that human cell extracts can catalyze error-free repair of CAG/CTG hairpins in a nick-directed manner. The repair system specifically targets CAG/CTG tracts for incisions in the nicked DNA strand, followed by DNA resynthesis using the continuous strand as a template, thereby ensuring CAG/CTG stability. Proliferating cell nuclear antigen (PCNA) is required for the incision step of the hairpin removal, which uses distinct endonuclease activities for individual CAG/CTG hairpins depending on their strand locations and/or secondary structures. We discuss the implications of these data for understanding the etiology of neurological diseases and trinucleotide repeat instability.

Mismatch Recognition Protein MutSβ Does Not Hijack (CAG)n Hairpin Repair in Vitro

CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)n expansion is caused by abnormal binding of the MutSβ mismatch recognition protein to (CAG)n hairpins, leading to hijacking mismatch repair function during (CAG)n hairpin repair. We demonstrate here that MutSβ displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)n hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSβ does not inhibit (CAG)n hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSβ is involved in CAG repeat instability in humans.