The role of MutY homolog (Myh1) in controlling the histone deacetylase Hst4 in the fission yeast Schizosaccharomyces pombe.

The DNA glycosylase MutY homolog (Myh1) excises adenines misincorporated opposite guanines or 7,8-dihydro-8-oxo-guanines on DNA by base excision repair thereby preventing G:C to T:A mutations. Schizosaccharomyces pombe (Sp) Hst4 is an NAD(+)-dependent histone/protein deacetylase involved in gene silencing and maintaining genomic integrity. Hst4 regulates deacetylation of histone 3 Lys56 at the entry and exit points of the nucleosome core particle. Here, we demonstrate that the hst4 mutant is more sensitive to H(2)O(2) than wild-type cells. H(2)O(2) treatment results in an SpMyh1-dependent decrease in SpHst4 protein level and hyperacetylation of histone 3 Lys56. Furthermore, SpHst4 interacts with SpMyh1 and the cell cycle checkpoint Rad9-Rad1-Hus1 (9-1-1) complex. SpHst4, SpMyh1, and SpHus1 are physically bound to telomeres. Following oxidative stress, there is an increase in the telomeric association of SpMyh1. Conversely, the telomeric association of spHst4 is decreased. Deletion of SpMyh1 strongly abrogated telomeric association of SpHst4 and SpHus1. However, telomeric association of SpMyh1 is enhanced in hst4Δ cells in the presence of chronic DNA damage. These results suggest that SpMyh1 repair regulates the functions of SpHst4 and the 9-1-1 complex in maintaining genomic stability.

A structural hinge in eukaryotic MutY homologues mediates catalytic activity and Rad9-Rad1-Hus1 checkpoint complex interactions.

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase.

Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9-Rad1-Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67-110 of hTDG, and Val74 of hTDG plays an important role in the TDG-Hus1 interaction. In contrast to the core domain of hTDG (residues 110-308), hTDG(67-308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.

Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9-Rad1-Hus1.

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

MutY and MutY homologs (MYH) in genome maintenance.

The base excision repair carried out by bacterial MutY DNA glycosylase and eukaryotic MutY homolog (MYH) is responsible for removing adenines misincorporated into DNA opposite G and 7,8-dihydro-8-oxo-guanines (8-oxoG); thereby preventing G:C to T:A mutations. Escherichia coli MutY (EcMutY) can also remove adenines from A/C and A/5-hydroxyuracil and can remove guanines from G/8-oxoG mismatches at reduced rates. Thus, MutY has a minor role in reducing the mutagenic effects on G:C to A:T transitions and G:C to C:G transversions. The eukaryotic MYH can excise adenines misincorporated opposite GO, G, or C; remove 2-hydroxyadenines mispaired with A,G, and GO; excise G from G/GO mismatch weakly, thereby preventing G:C to T:A transversions. The in vitro and in vivo activities of MYH can be modulated by several proteins including apurinic/apyrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and mismatch recognition enzymes MSH2/MSH6. Recently, MYH has been shown to associate with the checkpoint proteins, Rad9, Rad1, and Hus1 (referred as the 9-1-1 complex). Thus, MYH-mediated base excision repair is coordinated with mismatch repair, DNA replication, cell-cycle progression, and DNA-damage checkpoints. Biallelic germ-line mutations in the human MYH gene are associated with recessive inheritance of multiple colorectal adenomas and carcinoma. MYH mutations can cause G:C to T:A mutations of the adenomatous polyposis coli (APC), K-ras, and other genes that control cellular proliferation in the colon.

Interaction of checkpoint proteins Hus1/Rad1/Rad9 with DNA base excision repair enzyme MutY homolog in fission yeast, Schizosaccharomyces pombe.

The DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated opposite DNA strands containing guanine or 7,8-dihydro-8-oxoguanine by base excision repair thereby preventing G:C to T:A mutations. MYH has been shown to interact with the proliferating cell nuclear antigen (PCNA) in both human and fission yeast Schizosaccharomyces pombe systems. Here we show that S. pombe (Sp) MYH physically interacts with all subunits of the PCNA-like checkpoint protein heterotrimer, SpRad9/SpRad1/SpHus1, in yeast extracts and when the individual subunits are expressed in bacteria. The SpHus1 and SpPCNA binding sites are located in discrete regions of SpMYH. Immunoprecipitation assays reveal that the interaction between SpHus1 and SpMYH increases dramatically after hydrogen peroxide treatment, and this increase in the SpHus1-SpMYH interaction correlates with the presence of SpHus1 phosphorylation. In contrast, the interaction between SpPCNA and SpMYH after hydrogen peroxide treatment remains nearly unchanged. SpMYH associates with SpHus1 in a complex of approximately 450 kDa, the reported native molecular mass of the SpRad9/SpRad1/SpHus1-MYC complex. A larger portion of SpMYH shifts to the 150-500-kDa regions after hydrogen peroxide treatment in comparison with untreated extracts. SpHus1 phosphorylation is substantially reduced in SpMYH Delta cells after hydrogen peroxide treatment. These data suggest that MYH may act as an adaptor to recruit checkpoint proteins to the DNA lesions.

A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction.

HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC. The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase. The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self). In addition, matches were found to gp55 (T4 late sigma factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein). Abundant amino acid sequence matches were found to aa region 118-134 of gp55. Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111-136. Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17-gp55 interaction in regulation of terminase or other late gene transcription. However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to approximately 10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently. Moreover, gp55 residues 111-136 peptide specifically blocked DNA packaging in vitro. These results suggest that the T4 terminase interaction with T4 late sigma factor gp55 plays a role in DNA packaging in vivo. The gp55 interaction may function to load the terminase onto DNA for packaging.

Functional interaction of MutY homolog with proliferating cell nuclear antigen in fission yeast, Schizosaccharomyces pombe.

The MutY homolog (MYH) is responsible for removing adenines misincorporated on a template DNA strand containing G or 7,8-dihydro-8-oxoguanine (8-oxoG) and thus preventing G:C to T:A mutations. Human MYH has been shown to interact physically with human proliferating cell nuclear antigen (hPCNA). Here, we report that a similar interaction between SpMYH and SpPCNA occurs in the fission yeast Schizosaccharomyces pombe. Binding of SpMYH to SpPCNA was not observed when phenylalanine 444 in the PCNA binding motif of SpMYH was replaced with alanine. The F444A mutant of SpMYH expressed in yeast cells had normal adenine glycosylase and DNA binding activities. However, expression of this mutant form of SpMYH in a SpMYHDelta cell could not reduce the mutation frequency of the cell to the normal level. Moreover, SpMYH interacted with hPCNA, and SpPCNA interacted with hMYH but not with F518A/F519A mutant hMYH containing mutations in its PCNA binding motif. Although the SpMYHDelta cells expressing hMYH had partially reduced mutation frequency, the F518A/F519A mutant hMYH could not reduce the mutation frequency of SpMYHDelta cells. Thus, the interaction between SpMYH and SpPCNA is important for SpMYH biological function in mutation avoidance.

Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6.

Adenines mismatched with guanines or 7,8-dihydro-8-oxo-deoxyguanines that arise through DNA replication errors can be repaired by either base excision repair or mismatch repair. The human MutY homolog (hMYH), a DNA glycosylase, removes adenines from these mismatches. Human MutS homologs, hMSH2/hMSH6 (hMutSalpha), bind to the mismatches and initiate the repair on the daughter DNA strands. Human MYH is physically associated with hMSH2/hMSH6 via the hMSH6 subunit. The interaction of hMutSalpha and hMYH is not observed in several mismatch repair-defective cell lines. The hMutSalpha binding site is mapped to amino acid residues 232-254 of hMYH, a region conserved in the MutY family. Moreover, the binding and glycosylase activities of hMYH with an A/7,8-dihydro-8-oxo-deoxyguanine mismatch are enhanced by hMutSalpha. These results suggest that protein-protein interactions may be a means by which hMYH repair and mismatch repair cooperate in reducing replicative errors caused by oxidized bases.