Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4380-4384, September 1979
Biochemistry
Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups (conserved viral genes/25I-labeled peptide mapping/DNA replication/human cells)
MAURICE GREEN*, KARL BRACKMANN*, WILLIAM S. M. WOLD*, MARIA CARTAS*, HELEN THORNTON*, AND JOHN H. ELDERt *Institute for Molecular Virology, St. Louis University School of Medicine, 3681 Park Avenue, St. Louis, Missouri 63110; and tDepartment of Cellular and
Developmental Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 Communicated by Frank J. Dixon, June 21, 1979
Proteins that are firmly associated with genomic DNAs or RNAs have recently been identified in viral and nonviral systems, including adenoviruses (Ad) (1-7), picornaviruses (8-10), small Bacillus bacteriophages (11-13), possibly papovaviruses (14, 15), ColEl and CoIE2 plasmids (16), replicating OX174 (17), and possibly Escherichia coli DNA (18, 19). The linkage between these nucleic acids and proteins is very likely covalent and, in some cases, this has been formally proved. Covalently bound proteins (CBPs) are of great interest because they are widely believed to function in DNA or RNA replication. In the Bacillus bacteriophage and picornavirus systems, these proteins appear to be a viral gene product involved in viral genome replication. How these proteins function is not known, and studies on this point should be important to the overall understanding of nucleic acid replication. Ads provide an excellent model to study the replication of linear duplex DNA molecules in eukaryotic cells (reviewed in refs. 20-22). Ad DNA replicates in an overall 5'-3' direction by a strand displacement mechanism, with initiation and termi-
nation occurring at or near both termini. At least three viral coded early proteins and almost certainly a variety of cell-coded proteins are involved in Ad DNA replication. There are at least 31 human Ad serotypes that fall into five DNA genome homology groups, A-E (23). Some of the properties of the Ad2 or Ad5 (both group C) CBPs have been documented, but relatively little is known about this important protein. It is bound to both 5' termini, it is "sticky" and causes DNA-protein complexes to coalesce (2-5, 24), and it enhances the transfection infectivity of Ad DNA (4, 25). The AdS protein has an apparent Mr of 55,000, as estimated by sodium dod~cyl sulfate (NaPodSO4)/polyacrylami4e gel electrophoresis of 151-labelpd CBP (5)The gqnetic origin of the Ad protein has not been established. In previous studies, we showed that Ad2 CBP is synthesized during early stages of infection (22, 26) and, therefore, could be a product of either an early Ad gene or a cellular gene. No relationship by two-dimensional tryptic [15S]Met-peptide mapping was found to the known Ad2-coded early polypeptides, including El (Mr 40,000-50,000), El (Mr 53,000), E2 (Mr, 73,000), E2 (Mr 47,000), and E2 (Mr 42,000) (27). [35S]Met-peptide maps of CBP were also unrelated to maps of any of the virion structural proteins (unpublished data). Thus, the protein probably corresponds to either an unidentified Ad early gene product or to a cell gene product. In this report we show that DNAs of all five groups of human Ads have CBPs of Mr 55,000. We have also compared the tryptic and chymotryptic peptide maps of CBPs representing the five groups. Because it is difficult to obtain workable quantities of the protein labeled with [35S]methionine, we have mapped CBP labeled in vitro with 125[ by the procedure of Elder et al. (28), which can reproducibly label microgram quantities of polypeptides in NaDodSO4/polyacrylamide gels for peptide mapping. Our results show that the '25I-labeled peptide maps of CBP from Ads in each group are very closely related, whereas the maps of several virion proteins differ significantly. The similarity in the CBP peptide maps is remarkable because the DNAs of Ads in the five groups are
Biochemistry
Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups (conserved viral genes/25I-labeled peptide mapping/DNA replication/human cells)
MAURICE GREEN*, KARL BRACKMANN*, WILLIAM S. M. WOLD*, MARIA CARTAS*, HELEN THORNTON*, AND JOHN H. ELDERt *Institute for Molecular Virology, St. Louis University School of Medicine, 3681 Park Avenue, St. Louis, Missouri 63110; and tDepartment of Cellular and
Developmental Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 Communicated by Frank J. Dixon, June 21, 1979
Proteins that are firmly associated with genomic DNAs or RNAs have recently been identified in viral and nonviral systems, including adenoviruses (Ad) (1-7), picornaviruses (8-10), small Bacillus bacteriophages (11-13), possibly papovaviruses (14, 15), ColEl and CoIE2 plasmids (16), replicating OX174 (17), and possibly Escherichia coli DNA (18, 19). The linkage between these nucleic acids and proteins is very likely covalent and, in some cases, this has been formally proved. Covalently bound proteins (CBPs) are of great interest because they are widely believed to function in DNA or RNA replication. In the Bacillus bacteriophage and picornavirus systems, these proteins appear to be a viral gene product involved in viral genome replication. How these proteins function is not known, and studies on this point should be important to the overall understanding of nucleic acid replication. Ads provide an excellent model to study the replication of linear duplex DNA molecules in eukaryotic cells (reviewed in refs. 20-22). Ad DNA replicates in an overall 5'-3' direction by a strand displacement mechanism, with initiation and termi-
nation occurring at or near both termini. At least three viral coded early proteins and almost certainly a variety of cell-coded proteins are involved in Ad DNA replication. There are at least 31 human Ad serotypes that fall into five DNA genome homology groups, A-E (23). Some of the properties of the Ad2 or Ad5 (both group C) CBPs have been documented, but relatively little is known about this important protein. It is bound to both 5' termini, it is "sticky" and causes DNA-protein complexes to coalesce (2-5, 24), and it enhances the transfection infectivity of Ad DNA (4, 25). The AdS protein has an apparent Mr of 55,000, as estimated by sodium dod~cyl sulfate (NaPodSO4)/polyacrylami4e gel electrophoresis of 151-labelpd CBP (5)The gqnetic origin of the Ad protein has not been established. In previous studies, we showed that Ad2 CBP is synthesized during early stages of infection (22, 26) and, therefore, could be a product of either an early Ad gene or a cellular gene. No relationship by two-dimensional tryptic [15S]Met-peptide mapping was found to the known Ad2-coded early polypeptides, including El (Mr 40,000-50,000), El (Mr 53,000), E2 (Mr, 73,000), E2 (Mr 47,000), and E2 (Mr 42,000) (27). [35S]Met-peptide maps of CBP were also unrelated to maps of any of the virion structural proteins (unpublished data). Thus, the protein probably corresponds to either an unidentified Ad early gene product or to a cell gene product. In this report we show that DNAs of all five groups of human Ads have CBPs of Mr 55,000. We have also compared the tryptic and chymotryptic peptide maps of CBPs representing the five groups. Because it is difficult to obtain workable quantities of the protein labeled with [35S]methionine, we have mapped CBP labeled in vitro with 125[ by the procedure of Elder et al. (28), which can reproducibly label microgram quantities of polypeptides in NaDodSO4/polyacrylamide gels for peptide mapping. Our results show that the '25I-labeled peptide maps of CBP from Ads in each group are very closely related, whereas the maps of several virion proteins differ significantly. The similarity in the CBP peptide maps is remarkable because the DNAs of Ads in the five groups are
