Journal article
The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding
Nucleic acids research, Vol.29(15), pp.3270-3276
08/01/2001
DOI: 10.1093/nar/29.15.3270
PMCID: PMC55822
PMID: 11470885
Abstract
Replication protein A (RPA) is a heterotrimeric, multi-functional
protein that binds single-stranded DNA (ssDNA) and is essential
for eukaryotic DNA metabolism. Using heteronuclear NMR methods we
have investigated the domain interactions and ssDNA binding of a
fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment
contains an N-terminal domain (NTD), which is important for hRPA70–protein interactions,
connected to a ssDNA-binding domain (SSB1) by a flexible linker
(hRPA70
1–326
). Correlation analysis of the amide
1
H
and
15
N chemical shifts was used to compare the structure
of the NTD and SSB1 in hRPA70
1–326
with two
smaller fragments that corresponded to the individual domains. High
correlation coefficients verified that the NTD and SSB1 maintained their
structures in hRPA70
1–326
, indicating weak interdomain
coupling. Weak interdomain coupling was also suggested by a comparison
of the transverse relaxation rates for hRPA70
1–326
and
one of the smaller hRPA70 fragments containing the NTD and the flexible
linker (hRPA70
1–168
). We also examined the structure
of hRPA70
1–326
after addition of three different
ssDNA substrates. Each of these substrates induced specific amide
1
H
and/or
15
N chemical shift changes in both the
NTD and SSB1. The NTD and SSB1 have similar topologies, leading
to the possibility that ssDNA binding induced the chemical shift
changes observed for the NTD. To test this hypothesis we monitored
the amide
1
H and
15
N chemical shift changes
of hRPA70
1–168
after addition of ssDNA. The
same amide
1
H and
15
N chemical shift changes
were observed for the NTD in hRPA70
1–168
and
hRPA70
1–326
. The NTD residues with the largest
amide
1
H and/or
15
N chemical shift
changes were localized to a basic cleft that is important for hRPA70–protein
interactions. Based on this relationship, and other available data,
we propose a model where binding between the NTD and ssDNA interferes
with hRPA70–protein interactions.
Details
- Title: Subtitle
- The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding
- Creators
- Gary W Daughdrill - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USAJennifer Ackerman - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USANancy G Isern - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USAMaria V Botuyan - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USACheryl Arrowsmith - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USAMarc S Wold - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USADavid F Lowry - Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USA
- Resource Type
- Journal article
- Publication Details
- Nucleic acids research, Vol.29(15), pp.3270-3276
- Publisher
- Oxford University Press; Oxford, UK
- DOI
- 10.1093/nar/29.15.3270
- PMID
- 11470885
- PMCID
- PMC55822
- ISSN
- 0305-1048
- eISSN
- 1362-4962
- Language
- English
- Date published
- 08/01/2001
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984025289602771
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