PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX

LBL-11251 Preprint c-.r Lawrence Berkeley Laboratory UNIVERSITY OF CALIFORNIA I:' Submitted to the Journal of Biological Chemistry· PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX Rae Lyn Burke, Bruce M. Alberts, and Junko Hosoda July 1980 TWO-WEEK LOAN COpy This is a Library Circula tin 9 Copy which may be borrDwed for two weeks .. For a personal retention copy, call Tech. Info. Dioision, Ext. 6782 Prepared for the U.S. Department of Energy under Contract W-7405-ENG-48


TWO-WEEK LOAN COpy
This is a Library Circula tin 9 Copy which may be borrDwed for two weeks .. .i.cation system, ",'hile leav othe:LEl 1) Like intact 32 protein the 32 Th e r ication complex reconstructed from these proteins c resembles that formed in vivo. For the isolation of mutants in each of these prote1ns with major defects 1n T4 DNA is indicates that each of these proteins has a central role in DNA (3,4). Moreover, these proteins coordina function in vitro to propagate a fork on lex at a rate (2, slmilar to that measured 1n vivo (6) .,vhile an of correct base insertion (7,8). , T7 hac to (16)  protein or 32*1 protein was used at 100 Ug/m1. The reaction was incubated at 30 D e, and aliquots were removed and spotted onto DEAE paper (Whatman DE-81) and washed as described (37). The filters were dried and counted in a toluene-based 1 scintillation cocktail.
fhemica~~ -Enzyme grade sucrose was obtained from Schwartz-Marmo Ribo-and deoxyribonucleotides were purchased from Sigma, and the radiolabeled nucleotides were purchased from Amersham.
The reaction products were spread by a modified Kle.insmid t tec.hnique directly from a formamide hypophase onto a carbon-coated copper grid without prior deproteinization, as previously described (38). After shadowing with platinum. these grids were examined in a Philips EM-300 electron microscope.  In marked contrast to the normal five protein reaction, the five-  protein and nucleotide substrates, such a preincubation has no effect on the control reaction employing intact 32 protein.

RESULTS
In a similar experiment, a restriction fragment-primed, singlestranded DNA circle (r/Jx 174 DNA primed by the Hae III Z2 fragment) was used as the template (Fig. 4b). On this template, the DNA polymerase alone synthesizes a product equivalent to copying 20% of the available single-  (Fig, 7a) (44). To test whether the removal of the A peptide from 32 protein alters its intrinsic affinity for LtJ protein, a mixture of the DNA and either intact 32 protein or 32*I was sedimented through sucrose gradients. As shown in . 8, the T4 DNA polymerase alone sediments as a peak which moves more slO\,fly than an alkaline phosphatase marker (Fig, 8a), while both the intact 32 (Fig, Be) and the 32*1 protein (Fig. 8b) self·-associate and therefore sediment across a broad region of the :Lent, The sedimentation rate of the DNA polymerase is increased dramatically in the presence of 32 protein, and it now cosediments with the bulk of the 32 protein oligomeric complex ( Fig. 8e and d).
However, when co-sedimented with 32*1 , the DNA polymerase sediments at a rate which is indistinguishable from the rate measured in the absence of 32 protein (compare 8a and 8b). Thus, the rrnnoval of the carboxyl terminus from 32 protein has reduced its affini for the if ~l protein to less than 10 M , the limit of detection this assay. (ll,5).  (24,25).
Although the A domain is not required for cooperative interactions, its conformation changes upon cooperative DNA binding, since it becomes more readily digestible by chymotrypsin (21,22), as well as newly susceptible to staphyloccal protease cleavage at three discrete sites (47),7 Although their cooperative DNA interactions are very similar, the 32*1 DNA complex is more compact than the 32 protein complex. as indicated both by our sedimentation studies (Fig. 8)  We propose that the helix-invasion potential of 32 protein must be controlled to random, widespread denaturation of intracellular DNA and that this is why such invasion is normally blocked by the A domain.
However, it is tempting to speculate that a 32*I-like ae is functional ahead of a replication fork, ,vhere special protein-protein interactions It has been shown that 32 protein interacts s with 61 protein in the absence of DNA. These two proteins cosecliment dur sucrose ient sedimentation and coelute upon filtration chromatography. '1 No such association was found between 32*1 protein and 61 protein. control via interactions at its A domain is presented in Fig, 9.
As an aside, it is possible that H-D proteins in some replication systems may be designed to block DNA is at the level of priming.
In parti,cular, adenovirus DNA replication proceeds via leading-strand synthesis only, lagging-strand synthesis being completely blocked (50), the 72,000 dalton adeno-2 DNA binding protein (19),    DNA circles as template. The reaction was incubated for 5 min at 30 D C and stopped by the addition of Na 3 EDTA to 10 mM and NaOH to 6 mH final concentrations. The DNA was spread by a modified Kleinsmidt technique (38) and viewed with a Phillips Hodel 300 electron microscope . and 100 llg/ml bovine serum albumin) was over on a 5 ml preformed 5~30% (w/v) linear sucrose gradient in the same buffer and The significance of this result is unclear, as the A peptide obtained may have been denatured during isolation (it could be impossible to obtain "native" A peptide if the most stable conformation of the isolated fragment differs substantially from that of the covalently bound A peptide region).

Fi~.
Schematic view of some 32 protein interactions.
I. Favorable electrostatic contacts between the core region of 32 protein and the phosphates of the DNA backbone are postulated to    . , " This report was done with support from the United States Energy Research and Development Administration. Any conclusions or opinions expressed in this report represent solely those of the author(s) and not necessarily those of The Regents' of the University of California, the Lawrence Berkeley Laboratory or the United States Energy Research and Development Administration. ) .