The specific interactions of HMG 1 and 2 with negatively supercoiled DNA are modulated by their acidic C-terminal domains and involve cysteine residues in their HMG 1/2 boxes.
Sedimentation and gel retardation studies show a stronger interaction of HMG 1 and 2 with negatively supercoiled DNA than with linear, nicked-circular, or positively supercoiled ds-DNA. An apparent unwinding angle of 58 degrees was obtained for HMG 1 and 2 when assayed by protection of negatively supercoiled DNA from topoisomerase I relaxation or when assayed by the supercoiling of nicked-circular DNA with T4 DNA ligase. The protection of negatively supercoiled DNA was linear up to molar ratios of about 250:1. There was little change in binding reactions or in the protection of supercoiled DNA at ratios above 250:1, indicating that both activities saturate and that HMG 1 and 2 have binding site sizes of about 20 bp. P1, the major tryptic fragment of HMG 1 or 2 which retains the two DNA binding HMG 1/2 boxes, displays a 2-fold increase in binding to all types of ds-DNA compared to intact HMG 1 or 2. However P1 protects negatively supercoiled DNA from topoisomerase I relaxation about 5-fold less than intact HMG 1 or 2. Complete protection with P1 occurs at a molar ratio 1040:1, indicating a DNA binding site size of about 4 bp and an apparent unwinding angle of 10 degrees. P1 binding to closed-circular ss-DNA also involves a binding site of about 4 bp. Adding the acidic C-terminal fragment to P1 reversed its binding and allowed topoisomerase I to relax supercoiled DNA. These findings highlight the importance of the acidic C-terminal domains of HMG 1 and 2 in limiting electrostatic interactions of the HMG 1/2 boxes with ds- or ss-DNA. N-Ethylmaleimide inhibited the binding of intact HMG 1 or 2 to negatively supercoiled DNA, but did not inhibit the electrostatic binding of HMG 1 or 2 to ss-DNA, or of P1 to any form of DNA (ds or ss). These results suggest that cysteine residues are involved in the specific interaction of HMG 1 or 2 with negatively supercoiled DNA and that the acidic C-terminal domains modulate an intramolecular conformational change involving sulfhydryls within the HMG 1/2 boxes.
Sheflin
L G
LG
Department of Medicine, Buffalo Veterans Administration Medical Center, New York 14215.
Fucile
N W
NW
Spaulding
S W
SW
D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D013329:Structure-Activity Relationship
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D010446:Peptide Fragments; D003545:Cysteine; D005033:Ethylmaleimide