J. Mol. Biol. (1976) 101: 417425

LETTERS TO THE EDITOR

A Model for Replication Repair in Mammalian

Cells

A model for replication repair based on the process of branch migration explains the transient production of doubly substituted DNA within the first generation of incubation in bromodeoxyllridine and the appctarancc of four-pronged replication forks. DNA excision repair has been adequately demonstrated in mammalian cells (Regan et al., 1968,197l; Painter & Cleaver, 1969; Brandt et aE., 1972; Painter & Young. 1972), as has the second major recovery network, post-replication repair (Buhl & Regan, 1973; Lehmann, 1972). In contrast to bha bacterial mechanism in which a recombinational event occurs (Rupp et al., 1971), post, replication repair in mammalian cells appears to take place without recombination (Lehmann, 1972). A “gap-filling” model has been proposed to account for the ability of an organism to bypass lesions during DNA replication (Lehmann, 1972). This model supposes that normal synthesis proceeds until blocked by a lesion on one template strand. Replication of the complementary strand continues until a new initiation point is reached. At this point replication again occurs on both strands. The resulting single-stranded gap is filled in later. Although replication produces single-stranded regions opposite ultraviolet light,-induced lesions in bacteria (Iyer & Rupp, 1971; Ganesan, 1974), the evidence for the existence of such gaps in mammalian cells is not as convincing (Painter, 1974; Scudiero & Strauss, 1974). We wish to propose an alternative model which does not involve replication “gaps” and which makes use of the observed processes of strand displacement and branch migration (Masamune & Richardson, 1971; Lee et aZ., 1970; Broker & Lehman, 1971). Our model includes a mechanism for the replication of DNA past non-template regions : other hypotheses are reticent about the way the single-stranded gaps are “filled in”. The principal feature of our model is the use of a newly synthesized strand as an alternate template for the replication of a damaged region in the homologous parental strand, thereby providing a detour around a lesion which is not recognized as a functional template (Fig. 1). Alkylating compounds, ionizing and non-ionizing radiation produce lesions in DNS which inhibit synthesis. We assume that normal replicative synthesis proceeds until blocked by a lesion on one template strand (Fig. l(a)) and that replication of the complementary strand continues past the point of the lesion. Reassociation of parental strands by displacement of newly synthesized DNA (branch migration) permits pairing of the two daughter strands and provides an alternate template for synthesis (Fig. l(b)). The copying of the sequences complementary to the damaged region (Fig. l(c)) bridges the block to replication and allows synthesis to proceed without necessitating the removal of the initial lesion (Fig. l(d)). The model has been drawn so that bhere is a free 3’OH group to serve as primer on the alternate template. This formulation requires some unspecified factor to open up the helix at the growing point. The model can be drawn

N.

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STRAUSS

Branch migration

(b)

I

f

(c) Synthesis ___+

FIQ. 1. Model of replication template allowing replication

-------\ -eq--L; ------I

repair. Strand displacement to bypass a lesion (X).

and branch

I A

migration

create

an alternate

equally well with reversed DNA polarity but this form requires an initiation site for DNA synthesis on the alternate template. The model leads to at least two predictions: (1) since hydrogen bonding between the two displaced strands produces a duplex in which both strands are newly synthesized, DNA synthesized in the presence of BrdUrd will include shear-produced fragments of heavy density even in the first round of replication; (2) the displacement of newly synthesized strands should result in microscopically observable fourpronged replication structures in which one arm is substantially shorter than the other three (Fig. l(b) and (c)). DNA of heavy density is observed after short periods of incubation in BrdUrd and the proportion of material banding in this position increases after treatment with electrophilic reagents (Fig. 2). A neutral CsCl gradient of DNA obtained from

A~-_ I.80 P II 70

MMS- treated

Fraction

number

FIG. 2. Neutral CsCl gradients of DNA synthesized in the presence of BrdUrd after treatment of HEp.2 cells (a heteroploid cell line derived from a human carcinoma of the larynx) with MMS. Cells were incubated for 2 h with 16 +v-BrdUrd and 1 PM-FdUrd, treated for 1 h with 3 mix-MMS in the presence of BrdUrd and labeled for 1 h by addition of [3H]TdR (16.6 &i/ml; 50 Ci/mmol). Phenol-extracted DNA was sheared 3 times through a 22 gauge needle and centrifuged in C&l for 60 h at 30,000 revs/min in the SW 50.1 rotor of the Beckman L2 ultracentrifuge as described by Kato & Strauss (1974). -e-a--, 3H incorporation; ---

Intermediate

4.pronged molecules

forks scored

1 /so0

Methyl methanesulphonate. Acetoxy acetyl aminofluorene.

were diluted to yield grids containing 20 to 40 DNA molecules per grid square (150 mesh). At this concentration, two DNA molecules are rarely observed in the same field, thus greatly minimizing the possibility of overlap. Grids were scanned and scored as reported by Broker & Lehman (1971). When completely replicated DNA of hybrid density from treated cells was examined, only one branched structure was seen in 900 molecules of the diluted preparation (Table 2). Since we expect replicating forms to be concentrated in the intermediate density fraction of alkylated samples (Kato & Strauss, 1974), this control provides additional evidence for the identification of the branched structures as replication intermediates. Although previous data indicate that such structures should contain small regions of single-strandedness (Kato & Strauss, 1974), DNA was spread under conditions which do not allow good distinction between double and single-stranded molecules (Davis et al., 1971). Some of the branched molecules appear to have an “H” rather than an “X” configuration but the dimensions of the bar of the “H” are not comparable to reported structures of this type (e.g. Broker & Lehman. 1971). Since the short connecting bar is no longer than the width of a DNA molecule, it is likely to represent a small overlap which results when one arm falls back across the junction during grid preparation. We believe that the short fourt’h arm represent’s the displaced newly synthesized strands. Of the 32 four-pronged structures observed, 21 had shorter arms which were not obscured by neighboring molecules. The contour lengths of these molecules were measured to determine the distance over which branch migration had occurred. Measurement of contour lengths was based on microscope magnifications with no internal standard. Although shearing during preparation for CsCl gradient centrifugation might alter the relative length of the arms. molecules with a major component consisting of heavy DNA would not band in the intermediate density fraction (Kato & Strauss, 1974). The average length of the short arm is approximately 0.65 pm representing roughly 1900 nucleotide pairs. The longest segment observed was 1.64 pm (4800 nucleotide pairs), the shortest was 0.15 pm (440 nucleotide pairs) and

LETTERS

TO

THE

4” I

EDITOR

the median was 0.61 pm. Of the 21 molecules measured, 13 had short arms less than 2000 nucleotide pairs long and only three were greater than 3000 nucleotide pairs in length. It is likely that molecules having undergone only a short migration (less than 100 nucleotide pairs) would go undetected and therefore our measurements may be biased toward the longer structures. The possibility that random shear events have determined the identity of the short arm is minimized by comparison with the remaining three arms. Of ten molecules where all arms were fully extended and thus measurable, the average length of the segments (excluding the short arm) was 3.6 pm, the median was 2.9 pm with the longest segment observed being 8.9 ,um and the shortest 1.2 pm. If our model is correct, the heavy DNA (Figs 2 and 3) should be produced by shearing the short arm from branched DNA molecules (Fig. l(b) and (c)). It should be

100

300

zoo

.; \ m t 2

I00

Fracllon

number

FIG. 3. Neutral C&l density gradient of DNA synthesized in the presence of BrdUrd after AAAF treatment of a lymphoblastoid line derived from a patient with xeroderma pigmentosum. Cells were incubated for 2 h with BrdUrd and FdUrd as described in the legend to Fig. 2, treated for 1 h with 5 pg AAAF/ml, incubated for 2 h with BrdUrd and FdUrd and then labeled by the addition of [3H]TdR for 1 h as described in the legend to Fig. 2. AAAF, Acetoxy arc+1 aminofluorene.

noted that the proportion of counts appearing in the heavy fraction (relative bo those at an intermediate density) is completely determined by the extent of shearing during preparation and does not reflect in viva events. These fragments should be no larger than the short arms of the multi-forked molecules described above. Electron micrographs of fragments sedimenting in the heavy region of a CsCl gradient of DNA from xeroderma cells showed molecules ranging from 0.08 pm to l-66 pm with a median of 0.59 pm and a mode of O-68 pm (2000 base pairs) for the 28 molecules seen, as predicted (Fig. 5). The replication forks of both prokaryotes and eukaryotes contain single-stranded regions which undergo branch migration (Okazaki et al., 1968; Delius et aE., 1971: Kreipstein & Hogness, 1974). Although it has not been demonstrated that t#hese

422

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ii.

KATO

ASJ)

PIG. 4. Electron micrographs of 4.pronge(l ~cplication forks region of C&l gradients of DNA f mm alkylated cells. Grids spreading technique of Davis et al. (1971), stained with many1 (80 : 20). Arrows indicate the short 4th prong representing 1 pm. Magnification of insets showing branch points is 28,000

13. S’J’l{.IUSS

found in tho intermediate dfsnsitj were prepared using the nqucow acetate and shadowed with I’t/P~l displaced strantls. Scale indicatw Y

observations reflect in vivo reactions? the occurrence of branch migration over considerable distances in vitro (Kim et al., 1972) coupled with theoretical considerations of the flexibility of DNA molecules and the free energy of branch movement (Sigal & Alberts, 1972; Meselson & Radding, 1975) make the hypothesis of the in viva importance of branch migration attractive. The appearance of doubly substituted DNA during short pulses of RrdUrd and our micrographs of four-pronged forks support the view that branch migration has occurred in our preparations.

LETTERS

TO

THE

EDITOR

Vrc:. .5. Electron micrographs of fragments from the hwvy rcgiou of a CaCl gradient from xrrockrma cells. Details as in Fig. 4. Scalr inctirates 0.5 /urn.

423

of 1)iK.A

The model we propose is properly one of replplicdion repair in contrast to the process in bacteria which is indeed post-replication repair, since it occurs far from DXA growing points. According to our view, t,he fact that, branch migration can occur at, any growing point is demonstrated by the appearance of heavy DNA in control cells (.Fig. 2). Although we cannot determine the exact extent to which this process proceeds in vitro, the proportion of HH DNA in gradients of DNA from control cells (3 to 7% in 5 different experiments) along wit,h the measured estimattl

424

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KATO

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B. STRAISS

of the proportion of label in the vicinity of growing points (4Ooj,, Scudiero & Strauss. 1974) suggests that almost all of the replicating forks may have undergone branch migration. Blocks to replication in control cells which would generate “X” structures might be found at the ends of complebed replicons but it seems likely that the majorit) of these structures are generated in vitro. This fact does not diminish the possibilit’> of the occurrence of branch migrat,ion in vivo and in fact demonstrates the absence of any physical constraint, to the process. The increased proportion of DNA binding at heavy and intermediate densities after treatments which inhibit DiYA synthesis is supportive of our hypothesis. The model does.not account for “error prone” repair. However, t’here are several post replication repair mechanisms in bacteria and nob all of these are subject to error (Witkin & George, 1973; Radman, 1974; Sedgwick, 1975). The same may be the case for eukaryotic cells and the error prone mechanism may be quantitatively t,he least important. We wish to thank Dr Hewson Swift of the Department of Biology, as well as the Department of Biophysics, for generous use of electron microscope facilities. The comments of Dr N. Cozzarelli were extremely helpful in preparation of the manuscript. In addition, we wish to acknowledge the expert assist,ance of Gerald Grofman in preparing photographs for publication. This work was supported by grants from the National Institutes of Hcalt)h (GM 07816, CA-14599) and from the U.S. Energy Research and Development Administration (E(ll-1)2040). Two of us (N.P.H. and K.K.) were trainees of a genetics training program funded by the National Institut,es of Health (GM00090). N. P. H~atrr~ K. KATO-~ B. STRAUSS

Department of Microbiology The University of Chicago Chicago, Ill. 60637 Received

17 July

1975

REFERENCES Andrews, A. D., Robbins, J. J., Kraemer, K. H. & Buell, D. N. (1974). J. Nat. Cant. Inst. 53, 691-693. Brandt, W., Flamm, W. & Bernheim, N. (1972). Chem. BioE. Interactions, 5, 327-339. Broker, T. R. & Lehman, I. R. (1971). J. Mol. Biol. 60, 131-149. Buhl, S. & Regan, J. (1973). Mutat. Res. 18, 191-197. Davis, R. W., Simon, M. & Davidson, N. (1971). Methods in Enzymology, 21, 413-428. Delius, H., Howe, C. & Kozinski, A. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 3049-3053. Ganesan, A. (1974). J. Mol. BioZ. 87, 103-119. Iyer, V. & Rupp, W. (1971). Biochim. Biophys. Acta, 228, 117-126. Kato, K. & Strauss, B. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1969-1973. Kim, J., Sharp, P. & Davidson, N. (1972). Proc. Nat. Ad. Sci., U.S.A. 69, 1948-1952. Kreigstein, A. J. & Hogness, D. S. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 135-139. Lee, C. S., Davis, R. W. & Davidson, N. (1970). J. Mol. BioZ. 48, I- 22. Lohmann, A. (1972). J. Mol. BioZ. 66, 319-337. Masamune, Y. & Richardson, C. (1971). J. BioZ. Chem. 246, 2692-2701. Meselson, M. & Radding, C. (1975). Proc. Nat. Acad. Sk., U.S.A. 72, 355-361. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. & Sugino, A. (1968). Proc. Nut. Acad. Sci., U.S.A. 59, 598-605. Painter, R. B. (1974). Genetics, 78, 139- 148. Painter, R. & Cleaver, J. (1969). Radiat. Res. 37, 451-466. t Present address: Bard College, Anandale-on-Hudson,

New York

12604, U.S.A.

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Painter, R. & Young, B. (1972). Mutat. Res. 14, 225-235. Aspects of Mutagenesis (Prakash, L., Radman, M. (1974). Molecular and Environmental Sherman, F., Miller, M., Lawrmcc, C. & Tabrr, H., eds.), pp. 128-140, C. Thomas, Springfield. R egan, J., Trosko, J. & Carrier, W. (1968). Biophys. J. 8, 319-325. Regan, J., Setlow, R. & Ley, R. (1971). Proc. Nat. Acud. Sci., U.S.A. 68, 708-712. Rommrlarre, J., Faures-Miller, A. & Errera, M. (1974). J. -?foZ. Biol. 90, 491-508. Rupp, W., Wilde, C., Reno, D. & Howard-Flanders, P. (1971). .J. Mol. Biol. 61, 25 44. Scudicro, D. & Strauss, B. (1974). J. &IoZ. BioZ. 83, 17 -34. Scudirro, D., Henderson, E., Norin, A. & Strauss, B. (1975). Mufat. Res. 29, 473 488. Svdgwick, S. (1975). J. Bacter%oZ. 123, 154-161. Sigal, N. I% AlbertIs, B. (1972). .J. IzIoZ. BioZ. 71, 789-793. Witkin, E. R: George, D. (1973). Genetics, 73, 91 108.