BlOCHEMlCAi

Vol. 70, No. 3, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DNA POSTREPLICATION REPAIR GAP FILLING IN TEMPERATURE-SENSITIVE dnaG, dnaC, dnaA MUTANTS OF Escherichia coli

Robert Department

Received

Carey

Johnson

of Basic and Clinical Immunology and Microbiology Medical University of South Carolina Charleston, South Carolina 29401

March

25,1976

SUMMARY : Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type Escherichia coli to ultraviolet light are filled during reincubation. In this study the s, dnaC, and dnaA gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.

INTRODUCTION The closing after

exposure

Escherichia

repair

dnaE,

gap filling

(l-6).

The ability

study

requirements

I have for

DNA piece

of these

types

genes

in DNA size

to increase

the

size

demonstrates

continued

were

of DNA synthesis

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

can be studied

to examine

the e.

some role

on alkaline

of bacteria

sucrose

their

mutants

demonstrate

to W

gradients. after

repair additional

and DNA chain for

exrA

has been

of DNA synthesized

possible

coli

in postreplication

in gap filling

studied

791

that

exposure

with

DNA polymerase

postreplication

DNA initiation

gap filling. initiation

have after

of specific

of change

(W)

the hypothesis

in DNA synthesized

to W irradiation

In this

Copyright All rights

gene products

role

light

(DNA) synthesized

temnerature-sensitive

and ~

of a cell

exposure

possessing supports

by analysis

acid

to ultraviolet

evidence

The potential

assessed

short

strains

Current dnaB,

in deoxyribonucleic

of bacteria

coli

activity. recA,

of gaps

proficiency. unique

propagation

potential a specific

roles.

by Both

deficiency

Vol. 70, No. 3,1976

B1OCHEMICAL

at a non-permissive PC7, synthesize rounds

amounts

to high

to initiate study

have a role

MATERIALS

strains

PC7 (leu' dnaG308)

in KB media

thymine

--strr

were

provided

is

current

rounds probably

unable

of DNA (8).

-dnaC,

when

In this

or -dnaG gene products

gap filling.

and then

were

(9).

Postreplication

shift

from

the

for

lac---(thr---

by J. A. Wechsler

cells Cells

were were

growth,

same as those incubation

(7). were

grown then

for

either

temperatures

irradiation,

strr leu'

The bacteria

the washed

took

*-

and BT308

or nonpermissive

300C to 43'C

the gradient

-thi-

dnaC7);

(ZO$i).

at optimal

trifugation

leu-

3 x 108cells/ml.

Procedures

strain

BT308,

the &,

repair

=-

of [3H3 thymidine

activity.

from

subsequent

mutant,

and

to complete

in replication

if

CRT46 (thr--

to about

60 J/mm2 of W,

with

an ability

CRT46,

AND METHODS

--tonA

pulse

A third

to establish

mutants,

to initiate

fragments

in postreplication

--E. coli dnaA46);

(7).

(Okazaki)

attempted

with

an inability

temperatures

nascent

I have

but

RESEARCH COMMUNICATIONS

Two DNA initiation

of DNA consistent

of DNA synthesis

shifted

-strr

temperature.

AND BIOPHYSICAL

was between

ilv-

w-

&'

Strains

were

exposed

to

grown

a 10 or 5 minute iced

for

or reincubated

DNA synthesis sucrose

cen-

by Rupp and Howard-Flanders

was at 30°C or 43OC for in less

thi-

and alkaline

published

place

tonA

than

75X and 95X with

50 minutes.

15 seconds.

The

Recovery

no correlation

with

particular

or sample.

RESULTS Previous polymerases

studies I and III

use of alkaline of rates

sucrose

gene product

the conclusion

the possible

in postreplication

of gap filling

Thus an exrA with

have examined

that

gradient

repair

essential

roles

gap filling

(1,2).

zonal

centrifugation

for

can be reviewed

in several

publications

deficiency the cells

was analyzed with

792

the exrA

for

rate

deficiency

of The

determination (4,lO). of gap filling were

capable

(4)

BIOCHEMICAL

Vol. 70, No. 3, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

I

4

0

8

12 Frartion

16 no.

I

I

20

24

0

Figure 1: precipitable for initiat

Alkaline sucrose gradient sedimentation profiles of acidtemp rature sensitive DNA from --E. coli mutant PC? (dnac), (0); with [ 3 Hlthymidine on. (A) No W, 10 min. inc bation 3 10 min. incubation with 1%-]thym.idine (0). (B) W, 60 10 min.' incubation with 13H]thymidine, followed by 50 min. rei&bation in 2&nl of thymine at 30°C (0) or 43'C (0). sucrose gradient Figure 2: Alkaline precipitable DNA from --E. coli mutant as described in Fig. 1.

of partial

repair.

--E. coli

at 30°C and -dnaC'

and -dnaA-

of alkaline

sucrose

DNA showed creased

that,

to its

at 43OC for rate

cells

normal

size

50 minutes,

comparable In contrast

dnaG gene product 43'C

the

normal

(Figs.

demonstrated

1 and 2).

of repair

on irradiated

parental

50 minutes,DNA

When the

to increase

profiles

cells

were

to normal

was inincubated size

at a

seen at 30°C.

for

the dnaA and dnaC gene product,

an essential

gene product size

at 30°C for

the DNA was able

Sedimentation

at 43'C.

of DNA synthesized

to the results

thermolabile

DNA reached

when incubated

incubated

to the rate

profiles of acidThe procedure was

PC7 and CRT46 are -dnaC+ and -dnaA+ when incubated

gradients

for

sedimentation CRT46 (dnaA).

role

was dnaG-.

in 50 minutes,

but

793

in gap repair With

at 43'C

(Fig.

a 30°C reincubation, it

did

not.

the 3).

At the

:

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 3: Alkaline sucrose gradient precipitable DNA from --E. coli mutant as described in Fig. 1.

sedimentation BT308 (dnaG>.

profiles of acidThe procedure was

DISCUSSION The results completion incubated

be discerned

are not

that

by study

between

essential

species

were

role and/or

relevant using

-dnaA-

dnaA+ or dnaC+. for

post for

and limits

of the

inherent

or -dnaC-

repair

various

stop"

cell

double

was initially

mutant

794

might

polAdnaA. technique

in filling

did

of gaps

Enrichment

for

functions

methods.

described

temperature-sensitive

and cells

(1,2),

in the gradient

of replication.

synchrony

of

of gap structures.

I and III

of the gene products terminus

the extent

Thus the -dnaA and -dnaC

replication polymerases

in

to the -dnaA and -dnaC gene products'

The dnaG gene product or "quick

that

no difference

of a temperature-sensitive

the origin

be possible

"immediate"

cells

as proposed

cultures

analysis

eclipsed

molecular might

role,

the batch allow

demonstrated

at 30°C and phenotypically

Some overlapping

not

study

of gap filling

gene products

Also,

of this

as a DNAsynthesis mutation

(7).

More re-

such

Vol. 70, No. 3, 1976

cently

Bouchg,

probably

BIOCHEMICAL

Zechel,

initiates

and Kornberg

nascent

The dnaG gene product triphosphates

into

template.

In this

continous

replication

catalyses

study

it

only

gaps only

replication

fork.

100% absence for

conclusions

that

replication

forks

of bulk

DNA synthesized

studies

partial

filling

activity

with

of individual

a possible

only

phenotype

gaps is

post-

of the gaps.

The

the more recent

essential

to fill

completed

specificity

W-induced

the movement

on both

is

It

dis-

at the non-permissive

occurs

forks.

the dnaG-

proposed

for

supports

UV irradiation

measure

suggested

repair

replication

DNA replicating

DNA as a

studies

opposite

The dnaG gene product after

stranded

early

replication.

ribonucleotide

50% of the postreplication

of postreplication

(11).

four

of DNA synthesis

involve

discontinuous

of the gaps remaining

that

DNA synthesized

would

single

dnaG gene product chromosome

of the

This

initiation

the

in --E. coli

using

on one strand. for

that

the incorporation

the dnaG gene product

gaps on both gradient

fragments

was of interest

within

This

temperature

sucrose

reported

an oligoribonucleotide

replication

repair

have

(Gkazaki)

of the dnaG gene product

apparent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

arms of DNA for

the joining

the postreplication

should

be noted

gap filling. generated

that Further

at 43'C

for

alkaline analysis any

in progress.

ACKNOWLEDGMENTS This research was supported by the National Institute of General Medical I thank W. D. Rupp and S. Sedgwick for helpful disSciences (GM 18504). The excellent technical assistance of E. Terry is gratefully cussions. acknowledged. This is publication no. 28 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Tait, R.C., Harris, A.L. and Smith, D.W. (1974) Proc. Nat. Acad. Sci. U.S.A. 71, 675-679. Sedgwick, S.G. and Bridges, S.A. (1974) Nature 249, 348-349. Smith, K.C. and Meun, D.H.C.(1970) .I. Mol. Biol. 51, 459-472. Youngs, D.A., and Smith, K.C. (1973) J. Bacterial. 116, 175-182. Johnson, R.C. (1974) in Molecular Mechanisms for the Repair of DNA, pp. 325-329. Plenum Press, New York. Tomlin, N.V., and Svetlova, M.P. (1974) FBBS Letters. 43,185-188. Wechsler, J.A. and Gross, J.D. (1971) Mol. Gen. Genet. 113, 273-284.

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Vol. 70, No. 3, 1976

8. 9. 10. 11.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Bouche', J., Zechel, K. and Kornberg, A. (1975) J. of Biol. Chem. 250, 5995-6001. Rupp, W.D. and Howard-Flanders, P. (1971) Methods Enzymol. 21, 237-244. Sedgwick, S.G. (1975) J. Bacterial. 123, 154-161. Kurosawa, Y. and Okazaki, R. (1975) J. Mol. Biol. 94, 229-241.

796