Properties of DnaB helicase in [lambda] DNA replication

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 1991

OSTI ID:7176638

Stephens, K M

A tailed nicked-circle DNA substrate was used to measure the rapid replication fork (RF) movement catalyzed by E. Coli DnaB helicase and DNA polymerase III holoenzyme (pol III HE) (DnaB-RFs) (30 DnaB hexamers/substrate). The DnaB RFs can efficiently utilize the DNA substrate (60% in 5 min at 30C), and the forks move at a rapid rate (550-780 bp/sec at 30C). The DnaB-RFs have an average maximal processivity of 40,000 nt, and addition of either SSB or primase increase the processivity (150,000 nt + SSB, 70,000-140,000 nt + primase). However, SSB and primase do not affect the rate of fork movement or the amount of substrate utilized in the assay. The [lambda] SS proteins are effective at transferring DnaB onto the DNA substrate (8 DnaB hexamers/substrate). The [lambda] SS proteins do not change the rate of RF movement or the amount of substrate utilized. However, the amount of synthesis measured in the assay is [approximately]2-fold higher in the presence of the [lambda] SS proteins. Therefore, the [lambda] SS proteins increase the processivity of DnaB at the RF (100,000 nt). The [lambda] SS proteins do not appear to play a role in elongation because the processivity of the RF in the presence of SSB and primase is equivalent to the processivity of the [lambda] SS-RFs. [lambda] P protein blocks DnaB helicase activity if added to the RF assay prior to initiation or during elongation. DnaB helicase is more resistant to P inhibition, if the helicase is allowed to bind to the substrate prior to addition of [lambda] P or if primase and rNTPs are included in the assay. These results suggest that the conformation of the RF complex (DNA or nucleoprotein structure) blocks the attack of P on DnaB helicase. The heat shock proteins may play an auxiliary role in mediating the effects of [lambda] P if the concentration of P protein in the cells are high.

• Research Organization: Johns Hopkins Univ., Baltimore, MD (United States)
• OSTI ID: 7176638
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CHEMICAL PROPERTIES
DNA HELICASES
DNA POLYMERASES
DNA REPLICATION
ENZYMES
ESCHERICHIA COLI
KINETICS
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
REACTION KINETICS
TRANSFERASES