Replication Factors MCM2 and ORC1 Interact with the Histone Acetyltransferase HBO1

The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. We used a yeast two-hybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication. The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. We used a yeast two-hybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication. origin recognition complex minichromosome maintenance histone acetyltransferase bound to ORC1 pre-replicative complex histone acetyltransferase monocytic leukemia zinc finger protein MOZ-related factor HIV Tat-interacting protein human immunodeficiency virus males absent on the first something about silencing essential SAS-related acetyltransferase glutathione S-transferase Gal4 activation domain Gal4 DNA-binding domain multiplicity of infection 5-fluoroorotic acid hemagglutinin polymerase chain reaction dithiothreitol phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis amino acids 1,4-piperazinediethanesulfonic acid nucleotide Eukaryotic DNA replication is a tightly regulated process that is strictly coupled to cell cycle progression, ensuring that DNA is replicated only during S phase and that each origin is used only once per cell cycle. 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MYST proteins characterized by a highly conserved amino acid domain that of an C2HC zinc finger and a putative histone acetyltransferase domain. this we that MCM2 with HBO1 both in vitro and in and that this interaction is conserved in we the of both MCM2 and HBO1 and a role for the C2HC zinc finger of HBO1 in this We identify an allele of as as a suppressor mutation in HBO1 that restores the interaction with the defective MCM2. that two components of the MCM2 and ORC1, with a putative histone acetyltransferase and suggest a direct role for HAT activity in the process of DNA replication. 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A. PubMed Scopus Google Scholar) were as J. L. 1997; PubMed Scopus Google Scholar) and by two-hybrid A. D. M. R. K. in Full Text Full Text PDF PubMed Scopus Google Scholar). HBO1 was to with human and activity in a M. M. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HBO1 is a acid protein with a of and a N-terminal in and a conserved acid domain of the MYST family of proteins J. R. C. I. D. S. Genet. 1996; PubMed Scopus Google Scholar). The MYST domain a putative A domain and an C2HC zinc finger. the MYST HBO1 is highly conserved identical and to MYST family proteins the HBO1 interaction with we as a Gal4 activation domain and binding to the in the two-hybrid screen a HBO1 a N-terminal of HBO1 was a human and to in the of with of and MCM2 as as human and ORC1 in activation of the gene and of the with to activity with the and proteins. this may be to an of the protein to to and in this may the of an domain located in the N-terminal of HBO1 M. M. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This N-terminal activity may interactions in a as the yeast two-hybrid and may or with We to the binding in the yeast two-hybrid and to HBO1 is of with MCM2 and this we performed with in vitro HBO1 protein and proteins and in a and of protein was for the and with a for findings that HBO1 is of with MCM2 and ORC1 in that the interactions of HBO1 with MCM2 and ORC1 the interactions of HBO1 with MCM2 and ORC1 we N-terminal of human HBO1 N-terminal MCM2 and human ORC1 were with and as and cell were and with the The were for the of HBO1 or MCM2 by with or HBO1 be with human MCM2 that the interaction is conserved with human MCM2 in This interaction was infection of the and the of in the a MCM2 protein with HBO1 This interaction is the of MCM2 and as as we confirmed the interaction between human ORC1 and HBO1 M. Stillman B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) ORC1 and HBO1 Taken together, these the of a conserved interaction between MCM2 and HBO1 in and that HBO1 protein is of with both MCM2 and on findings that HBO1 with two components of the pre-RC in vitro and in and the that HBO1-associated HAT activity may be for the of histone we to the of HBO1 we with and and by M. M. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), revealed that HBO1 protein is to the MCM2 protein is both in the and we that a of the HBO1 protein is with a and that HBO1 is to by The of HBO1 protein is of a that human is to by A. M. M. R. Biol. Chem. 379: Google Scholar). The that HBO1 protein is and with a is with a role in regulation of the of the of HBO1 and to be We that HBO1 with MCM2 both in vitro and in vivo. the that the interaction between MCM2 and HBO1 is mediated by ORC1 or that the interaction is mediated by MCM2. we the interaction of HBO1 and MCM2 to these The highly conserved MYST domain of HBO1 is by the of two an C2HC zinc finger and an of the MYST family A. M. A. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) to the zinc of zinc finger Y. 1996; PubMed Scopus Google Scholar). C2HC zinc finger and E. M. Dev. Biol. PubMed Scopus Google Scholar), or as the first or of a of zinc A. H. J. PubMed Scopus Google Scholar, K. Cell. Full Text PDF PubMed Scopus Google Scholar). 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This is with the that of the of the zinc finger the of HBO1 to with MCM2 and ORC1 and the that the C2HC zinc finger of HBO1 a role in direct interactions with MCM2. The MCM family of proteins conserved in and essential for DNA replication. the precise of in DNA replication to be this we identified a interaction between the MCM2 and HBO1, a putative histone acetyltransferase and MYST domain together with the characterized interaction between ORC1 and HBO1 M. Stillman B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), suggest that human HBO1 protein may play a direct role in DNA replication. of the that the interaction between MCM2 and HBO1 is and the interaction was in yeast and in human we of and HBO1 that defective in to to we a direct interaction between MCM2 and HBO1 by a mutant of HBO1 that restores the to with This suppressor mutation as genetic of a direct interaction between MCM2 and HBO1. 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This that of is involved in the regulation of DNA replication in with the that HBO1 with two components of the replication MCM2 and ORC1, to that HBO1-associated HAT activity may play a direct role in of DNA replication. the and for interactions that a histone to a pre-replication complex, this only a that be in the of We the to this is a genetic that interaction with function. this of and MCM2 and that defective for interaction with HBO1 an to this This allele then be with for the to a in the gene or for the to in an in the this then be to isolate a suppressor as that restores the of the allele to This suppressor then be used to the the a between HAT activity and origin is an and that by is a in this We to for and this to for and to for in the of the