Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Sheflin LG,Fucile NW,Spaulding SWdoi
10.1021/bi00064a005subject
Has Abstractpub_date
1993-04-06 00:00:00pages
3238-48issue
13eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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