Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro

Steve M. Patrick, John Turchi

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin- damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6- fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.

Original languageEnglish (US)
Pages (from-to)8808-8815
Number of pages8
JournalBiochemistry
Volume37
Issue number24
DOIs
StatePublished - Jun 16 1998
Externally publishedYes

Fingerprint

Cisplatin
DNA
In Vitro Techniques
human RPA1 protein
Assays
DNA Repair
Proteins
Repair
Magnesium Chloride
Affinity chromatography
DNA Probes
DNA-Binding Proteins
Electrophoretic Mobility Shift Assay
Electrophoretic mobility
Cell Extracts
Affinity Chromatography
Protein Binding
Human Activities
Fractionation
Molecular Weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro. / Patrick, Steve M.; Turchi, John.

In: Biochemistry, Vol. 37, No. 24, 16.06.1998, p. 8808-8815.

Research output: Contribution to journalArticle

@article{b109d26e8d8c4944b511da91911fde9b,
title = "Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro",
abstract = "Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin- damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6- fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.",
author = "Patrick, {Steve M.} and John Turchi",
year = "1998",
month = "6",
day = "16",
doi = "10.1021/bi9730590",
language = "English (US)",
volume = "37",
pages = "8808--8815",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "24",

}

TY - JOUR

T1 - Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro

AU - Patrick, Steve M.

AU - Turchi, John

PY - 1998/6/16

Y1 - 1998/6/16

N2 - Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin- damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6- fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.

AB - Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin- damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6- fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.

UR - http://www.scopus.com/inward/record.url?scp=0032537491&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032537491&partnerID=8YFLogxK

U2 - 10.1021/bi9730590

DO - 10.1021/bi9730590

M3 - Article

C2 - 9628743

AN - SCOPUS:0032537491

VL - 37

SP - 8808

EP - 8815

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 24

ER -