Purification and characterization of nuclear factor III (origin recognition protein C), a sequence-specific DNA binding protein required for efficient initiation of adenovirus DNA replication.

E A O'Neill,T J Kelly

doi:10.1016/s0021-9258(19)35442-0

Abstract

Nuclear factor III (NF-III, origin recognition protein C) is a cellular DNA binding protein that has high affinity for a DNA sequence contained within the adenovirus origin of DNA replication. We have purified NF-III more than 760-fold from HeLa nuclear extracts by a combination of conventional methods and DNA recognition site affinity chromatography. The NF-III polypeptide has an apparent molecular weight of 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sedimentation coefficient of the native protein is 3.1 S, and the Stokes radius is 61 A. These values indicate that NF-III exists in solution as a somewhat asymmetric monomer. Purified NF-III binds specifically to a sequence within domain C of the adenovirus origin of replication and stimulates initiation of adenovirus DNA replication in in vitro reconstitution experiments. NF-III also binds specifically to a sequence element in the human histone H2B gene that is required for H2B-specific mRNA synthesis in vitro. Thus, NF-III may function as an activator of both viral DNA replication and cellular transcription.

Highlights

C) is a cellular DNA binding protein that has high within the first51 base pairs of the viral genome and encomaffinity for a DNA sequence contained within the ad- pass three separatedomains
NF-I11may function as an effect of NF-I on initiationis mediated via specific binding to activator ofboth viral DNA replication and cellular domain B of the adenovirus origin of DNA replication
Initiation of replication can within domain C has demonstrated the existence of a correoccur at either terminuosf the linear,35,000-bp’ viral genome lation between the replication efficiency of a given mutant.The initiation reaction consists of the formation of C/NF-111, suggesting a role for the protein iandenovirus DNA

Summary

EXPERIMENTAL PROCEDURES

Protein that is covalently attached to the 5’ termini of adenovirus genomic DNA; Ad pol, 140-koa DNA polymerase encoded by the Purification of NF-III adenovirus genome; NF-I, nuclear factor I; NF-111, nuclear factor 111; Buffers-Buffer H is 25 mM HEPES (pH 7.5), 5 mM KCI, 1 mM. The matrix was washed with 0.025 M NaCl in Buffer T (25 ml),and Renaturation of Gel-purified NF-IIZ-The peak fraction of NF-111 the DNA binding activity was eluted with a linear gradient from 0 to from the hydroxylapatite eluate was electrophoresed on an SDS-. With the following modifications: a poolof the peak fractions from the specific DNA affinity column (50 p l ) was diluted with 100 pl of 0.025 M HEPES (pH 7.5), 0.01% Nonidet P-40, 0.001 M DTT, 0.001 M EDTA, 0.1 mM PMSF, and layered onto a linear 15-35% glycerol gradient (4.8 mI) prepared in Buffer S containing 0.1 M NaCI. This step effectively removes higher affinity, nonspecific DNA binding proteins thawt ould

RESULTS

Hydroxylapatite

Findings

DISCUSSION