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Functional residues on the surface of the N-terminal domain of yeast Pms1.

ABSTRACT: Saccharomyces cerevisiae MutLalpha is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5A crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.

SUBMITTER: Arana ME

PROVIDER: S-EPMC2856611 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Functional residues on the surface of the N-terminal domain of yeast Pms1.

Arana Mercedes E ME Holmes Shannon F SF Fortune John M JM Moon Andrea F AF Pedersen Lars C LC Kunkel Thomas A TA

DNA repair 20100401 4

Saccharomyces cerevisiae MutLalpha is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5A crystal structure of yeast Pms1 ( ...[more]

PMID: 20138591

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Project description:UnlabelledThe arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding.ImportanceThe mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding.

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Functional residues on the surface of the N-terminal domain of yeast Pms1.

Publications

Functional residues on the surface of the N-terminal domain of yeast Pms1.

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