Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins.
Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.
Chastain
Paul D
PD
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Makhov
Alexander M
AM
Nossal
Nancy G
NG
Griffith
Jack
J
D017122:Bacteriophage T4 / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D018741:Replication Origin / Q000235:genetics*; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D014764:Viral Proteins; D019915:DNA Primase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4