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 (MAP). A key feature of MYH activity is its coordination with the 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 between the catalytic and 8-oxoG recognition-domains of hMYH (IDC, residues 295-350) 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 (SpMyh1) as a model system. In vitro studies of SpMyh1 identified residues I 261 and E 262 of the IDC (equivalent to V 315 and E 316 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 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.

BackgroundSIRT6, a member of the NAD+-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9–Rad1–Hus1 (9–1–1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes.ResultsWe show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9–1–1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres.ConclusionsAlthough SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0041-9) contains supplementary material, which is available to authorized users.

The malarial parasite continues to be one of the leading causes of death in many developing countries. With the development of resistance to the currently available treatments, the discovery of new therapeutics is imperative. Currently, the plasmepsin enzymes found in the food vacuole of the parasite are a chief target for drug development. Allophenylnorstatine-based compounds originally designed to inhibit HIV-1 protease have shown efficacy against all four plasmepsin enzymes found in the food vacuole of Plasmodium falciparum. In this study, the first crystal structure of P. malariae plasmepsin 4 (PmPM4) bound to the allophenylnorstatine-based compound KNI-764 is described at 3.3 Angstroms resolution. The PmPM4-inhibitor complex crystallized in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.9, b = 112.6, c = 90.4 Angstroms, with two molecules in the asymmetric unit related by a non-crystallographic symmetry operator. The structure was refined to a final R factor of 24.7%. The complex showed the inhibitor in an unexpected binding orientation with allophenylnorstatine occupying the S1' pocket. The P2 group was found outside the S2 pocket, wedged between the flap and a juxtaposed loop. Inhibition analysis of PmPM4 also suggests the potential for allophenylnorstatine-based compounds to be effective against all species of malaria infecting humans and for the future development of a broad-based inhibitor.

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.

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

Thymine DNA glycosylase (TDG) is an essential multifunctional enzyme involved in DNA base excision repair, DNA demethylation, and transcription regulation. TDG is the predominant enzyme to remove thymine from T/G mispair, which arises due to deamination of 5-methylcytosine at the CpG dinucleotide, thereby preventing C to T mutations. SIRT1 is a member of class III NAD+-dependent histone/protein deacetylases. In this study, we demonstrate that SIRT1 interacts with the residues 67–110 of human TDG (hTDG). In addition, SIRT1 enhances TDG glycosylase activity and deacetylates acetylated TDG. TDG acetylation weakens its interaction with SIRT1. Although acetylated TDG has reduced glycosylase activity toward T/G, 5-formylcytosine/G, and 5-carboxylcytosine/G, it has a stronger activity toward 5-fluorouracil/G substrate as compared to unmodified TDG. SIRT1 weakly stimulates acetylated hTDG activity toward T/G, 5-formylcytosine/G, and 5-carboxylcytosine/G as compared to control hTDG. Sirt1 knockout mouse embryonic fibroblast cells have higher levels of TDG expression and acetylation. The physical and functional interactions between SIRT1 and TDG may mediate DNA repair, gene expression, and FU-mediated cytotoxicity.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N- (residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91-266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91-266-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91-266-1-1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent of complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.