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PDBsum entry 2a1k
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DNA binding protein
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PDB id
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2a1k
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References listed in PDB file
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Key reference
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Title
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Structure and enzymatic properties of a chimeric bacteriophage rb69 DNA polymerase and single-Stranded DNA binding protein with increased processivity.
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Authors
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S.Sun,
L.Geng,
Y.Shamoo.
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Ref.
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Proteins, 2006,
65,
231-238.
[DOI no: ]
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PubMed id
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Abstract
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In vivo, replicative DNA polymerases are made more processive by their
interactions with accessory proteins at the replication fork. Single-stranded
DNA binding protein (SSB) is an essential protein that binds tightly and
cooperatively to single-stranded DNA during replication to remove adventitious
secondary structures and protect the exposed DNA from endogenous nucleases.
Using information from high resolution structures and biochemical data, we have
engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase
and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase
with its cognate SSB via a short six amino acid linker increases affinity for
primer-template DNA by sixfold and subsequently increases processivity by
sevenfold while maintaining fidelity. The crystal structure of this fusion
protein was solved by a combination of multiwavelength anomalous diffraction and
molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned
proximal to the N-terminal domain of RB69 DNA polymerase near the template
strand channel. The structural and biochemical data suggest that SSB
interactions with DNA polymerase are transient and flexible, consistent with
models of a dynamic replisome during elongation.
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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.
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Figure 5.
Figure 5. F6 structure. A. Composite omit map showing part of
the density for DNA polymerase in F6 at 1.5  level.
B. Ribbon diagram of F6 structure with a zinc atom bound by SSB
and a GDP bound by DNA polymerase. Color coding is based on
crystallographic temperature factors (B), with red representing
regions of higher B factors and blue representing regions of
lower B factors (B factor range 1-100). The linker and 12 amino
acids from the C-terminus of RB69 SSB core domain are highly
flexible and were not seen in the electron density region
connecting the C-terminus of SSB to N-terminus of DNA
polymerase. C. Surface charge distribution of F6.
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The above figures are
reprinted
by permission from John Wiley & Sons, Inc.:
Proteins
(2006,
65,
231-238)
copyright 2006.
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