Figure 1.
Figure 1. F6 displays higher processivity than does RB69 DNA
polymerase. Primers were 5 end
labeled ^32P 20-mers and are complementary to different regions
on M13mp18. Proteins were incubated for 2 min at 30°C with
annealed primer-template DNA and dNTPs. Reactions were initiated
by the addition of MgCl[2] and stopped by the addition of
EDTA/formamide loading buffer. Some of the reactions also have
500 g/mL
heparin to produce single turn-over kinetics. Reaction products
are separated on a 6% polyacrylamide-7M urea gel. Lane 1:
Reaction mixture without enzyme. Lane 2: Heparin was added
before F6 as a control to show that the fusion binds heparin and
is an effective single-turnover sink. Lane 3-15: Reactions with
different protein and protein combinations with/without heparin
at different time points showing that compared with RB69 DNA
polymerase, F6 has increased processivity. A. Reactions with P1
as primer. B. The primer P2 is complementary to a region on
M13mp18, where there is a major replication pause site (12 bp
hairpin structure) 105 nt downstream. As shown in Lane 3, F6 was
able to overcome this major pause site and continue DNA
synthesis, while all other protein and protein combinations
stopped.
The above figure is reprinted
by permission from John Wiley & Sons, Inc.:
Proteins
(2006,
65,
231-238)
copyright 2006.
end
labeled ^32P 20-mers and are complementary to different regions
on M13mp18. Proteins were incubated for 2 min at 30°C with
annealed primer-template DNA and dNTPs. Reactions were initiated
by the addition of MgCl[2] and stopped by the addition of
EDTA/formamide loading buffer. Some of the reactions also have
500 
g/mL
heparin to produce single turn-over kinetics. Reaction products
are separated on a 6% polyacrylamide-7M urea gel. Lane 1:
Reaction mixture without enzyme. Lane 2: Heparin was added
before F6 as a control to show that the fusion binds heparin and
is an effective single-turnover sink. Lane 3-15: Reactions with
different protein and protein combinations with/without heparin
at different time points showing that compared with RB69 DNA
polymerase, F6 has increased processivity. A. Reactions with P1
as primer. B. The primer P2 is complementary to a region on
M13mp18, where there is a major replication pause site (12 bp
hairpin structure) 105 nt downstream. As shown in Lane 3, F6 was
able to overcome this major pause site and continue DNA
synthesis, while all other protein and protein combinations
stopped.