reca-polv-supp1

8/2/2019 RecA-PolV-supp1

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Ev

olutionofTLSmodels

1985 2001 2005 2006 year

Bridges-Woodgate two-step

RecA transactivation of pol V

5

5

template

RecA-s

sDNA

transac

tivator

pol V

RecA

3

ADP ATP

5

pol V-RecA Cowcatcher

3pol III

UmuDC

RecA

pol V-RecA mutasome

UmuC

UmuD2RecA

ATP

3

Supplementary Figure 1

Evolving models of SOS mutagenesis targeted at DNA template lesions caused bytranslesion DNA synthesis (TLS). Prior to the discovery ofE. colipol V (UmuD2C), TLSwas proposed to occur in two steps, with pol III first inserting a nucleotide opposite a 5T ofa TT pyrimidine dimer (X), whereas insertion opposite the 3T by pol III required thepresence of UmuDC interacting with a RecA molecule located at the 3-proximal RecAfilament end, located adjacent to the site of the template lesion. Following the discovery ofpol V, data showed that assembly of a RecA nucleoprotein filament blocks pol V-catalyzedDNA synthesis on damaged and undamaged DNA. However, TLS can occur provided that

the filament is disassembled in a 3 to 5 direction by an oncoming pol V in the presence of

single-stranded binding protein. The disassembly of the RecA filament was viewed inanalogy to a locomotive cowcatcher, a large triangular piece of metal attached to the frontend of a locomotive that pushes cows (i.e., RecA) off the train tracks. A subsequentstudy showed that a cis RecA filament, one which assembles on a template strand beingcopied, blocked TLS, whereas TLS could occur in the absence ofcis-acting RecA filament.

Two non-filamentous types of interactions between RecA and pol V were identified, whichappeared to be both necessary and sufficient for TLS. The data in this paper show that polV activity on either damaged or undamaged DNA requires transactivation by RecA boundto a separate ssDNA molecule.

Supplementary Figures, Legends and Methods


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3 nt oh

0 1.2 1.9 2.2 3.0 %PU

0 0.2 1 1.5 2.5 pol V (M)


Pol V catalyzes weak primer extension in the absence of RecA on a 3nt overhang hairpin

DNA (20 nM).

LexA

C-term

N-term

0 2 7 15 time (min)

Supplementary Figure 3a A 6 nt oligomer is insufficient to activate RecA. RecA (4 M) cannot assemble on a short

6mer (160 nM) in the presence of continuously regenerated ATP. An activated RecAnucleoprotein filament, as defined by LexA cleavage capacity, does not assemble on short6 nt oligomers. Thus, LexA (4 M) cannot undergo autocleavage as shown by the absence

of the two autocleavage fragments (C-term and N-term). In contrast, see SupplementaryFig. 3, where autocleavage of LexA occurs on a 50 nt overhang hairpin to generate C-term and N-term fragments.b Transactivation of pol V-catalyzed synthesis on a hairpin DNA with a 3 nt templateoverhang does not occur with ssDNA (80mer) in the absence of RecA, ssDNA (6mer) inthe presence of RecA or dsDNA in the presence of RecA. A sketch of the DNA supplied intrans is shown at the right. RecA was added at a concentration of 4 M when present in

the reaction. Reactions were performed as described in Methods.

3 nt oh

+trans ds95mer

1.2

+

+

4

1.2

+

++

5

1.5

++

6

% PU+

trans 6mertrans 80mer

RecApol V

trans ds95mer + RecA

trans 6mer + RecA

trans 80mer 5

a b


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Supplementary Figure 4a Bona fide RecA filaments are formed on 50 nt oh hairpin DNA. RecA (4 M) assembled on a 50 ntoverhang hairpin (20 nM) in the presence of continuously regenerated ATP facilitates complete LexAautocleavage (4 M) within 7 min into 2 fragments (C-term and N-term). Thus a RecA nucleoprotein filament,

as defined by LexA cleavage capacity, is present on a 50 nt overhang hairpin.b Transactivation of pol V synthesis on undamaged DNA hairpin containing a 50 nt template overhangoccurs in the presence of a ssDNA 80mer present in trans in either the absence or presence of sliding

clamp/ clamp loading complex (lanes 5 and 6, respectively). Pol V synthesis, as measured as % primer

utilization (% PU) is virtually absent in the absence of transactivating ssDNA. ST denotes streptavidin-biotinblocks used to inhibit dissociation of the clamp.

50 nt oh

LexA

C-term

N-term

0 7 15 30 time (min)

Supplementary Figure 5Transactivation of pol V synthesis on double hairpin substrates containing a ssDNA 50 nt undamaged DNAgap (left gel) and a gap with an abasic lesion (right gel) in the presence and absence of/ complex. Pol V

synthesis is measured as % primer utilization (% PU).

++trans 80mer

61

++

+

6

7

+

+

2

5

+

+

3

2

++

+

4

54

+

+

5

0.3% PU

/RecA

+pol V

1

St

50 nt oh

St

St St

50 nt gapX

St St

50 nt gap

X

12 40 %TLS

++++trans 80mer

45

+

+

+

6

7

+

7

10

+

+

8

5

+

+

9

8

+

+

+

10

38

+

+

11

40

+

+

+

12

5

+

+

2

7

+

+

3

5

+

+

+

4

31

+

+

5

3% PU+

/RecA

+pol V1

a b


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Transactivation of pol V in the presence clamp. Transactivator substrates for RecA were12 nt oh hairpins, 50 nt oh hairpins, double hairpins containing a gap (50 nt) and circularDNA containing a 210 nt gap. Pol V synthesis is measured as % primer utilization (% PU).Note that a hairpin containing a 12 nt overhang is insufficient for transactivation (lane 1).

A hairpin containing a 50 nt overhang stimulates pol V in the presence of RecA to asimilar degree than a 50 nt gapped DNA (lane 2 and 3). A 30mer strand of DNA annealedto 240mer circular DNA, which creates a 210 nt long ssDNA gap, results in strongertransstimulation of pol V (lane4).

240 nt

trans circle + RecA

++trans circle44

+

+

+

6

4

+

+

2

4

+

+

+

3

4

+

+

4

38

+

+

5

2% PU+

/RecA

+pol V1


Pol V synthesis on an undamaged circular DNA template can be activated by RecAbound to unprimed circular ssDNA in trans in the presence and absence of/ complex.

Pol V synthesis is measured as % primer utilization (% PU).

StSt

trans HP12 + RecA12 nt oh

12 nt oh

St St

+trans 50 nt gap

27

+

3

+trans HP50

+trans gap circle

+trans HP12

56

+

4

217% PU+

++pol V, RecA, /

21

50 nt gap

St St

trans 50 nt gap + RecA trans gap circle + RecA

StSt

trans HP50 + RecA

50 nt oh


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RecA protein inhibits DNA synthesis on 12 nt overhang hairpin DNA, but not on 3ntoverhang hairpin DNA.Pol IV (10 nM) synthesis was measured in the presence of RecA and ATPS. A 12 nt

overhang provides enough space for RecA to nucleate on and presumably over the 3-OHto prevent extension at higher RecA concentrations. RecA does not inhibit synthesisreactions on a 3 nt overhang template. Since Pol V does not show appreciable synthesisactivity on hairpin DNA in the absence oftrans ssDNA, it cannot be used to study RecAinhibition of DNA synthesis on the hairpin p/t DNA. Therefore,pol IV was used as acontrol polymerase.

3 nt

5

12 nt

5

48.7 49.7 13.5 2.2 % PU

1

+

3

2

+

4

0.50RecA (M)

++Pol IV

21

76.1 72.6 77.5 78.2 % PU

1

+

3

2

+

4

0.50RecA (M)

++Pol IV

21


Kinetics of pol V-transactivation by RecA-ssDNA. Pol V-catalyzed primer utilization (PU)when copying a hairpin containing a 3 nt template overhang was measured in thepresence of ATPS and RecA (2 M) at varying trans ssDNA (36mer) concentrations.

Primer extension efficiency was measured as a function of time. These data were used tocalculate the dependence of primer extension velocity on trans DNA concentration asplotted in the manuscript (Fig. 2 b).

Time (min)

Primerutilization(%)

5 nM

0 trans 36mer

10 nM

40 nM

80 nM

160 nM

3 nt oh


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input after 10 20 40washes min min min

22.7 21.3 27.0 29.0 relative %

+RecA+ATPS

magnetic

beads

+DNA

magnet

Remove SN

Redissolve, wash 3 times,

load aliquot on gel,

incubate for x min, repeat


RecA is stably immobilized on DNA-beads for at least 40 min. A biotinylated 30 mer wasattached to Streptavidin coated magnetic beads and incubated with excess RecA and ATPSfor 10 min at 37 C. The supernatant (SN) containing unbound RecA and ATPS was

decanted after separation with a magnet. The RecA beads were washed and resuspended in

the buffer, and, after washing, an aliquot was removed for separation on a protein gel. Theremaining beads were incubated for up to 40 min at 37 C, separated to remove RecA thathad dissociated from the DNA, and resuspended in buffer. An aliquot was removed forseparation on a protein gel and subsequent Western blotting with antibodies against RecA.The bands were qualitatively compared using Imagequant software (Molecular Dynamics).Theamount of RecA on bead-DNA did not change significantly following a 40 min incubation.


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Wt 17/730 730 17 Wt

5

55

5

5 5

3

33

3

33

35

30ntB

RecA

19.0 23.2 15.3 20.0 22.6 relative %

35

30ntB

Wt 17/730 730 17 Wt

gprotein

0.58 0.55 0.56 0.73 0.570.1 0.05 0.1 0.01 0.2

RecA

Supplementary Figure 11The amounts of wild type (Wt) and mutant RecA on DNA-beads are comparable. A 30merbiotinylated near the 5 end was attached to Streptavidin coated magnetic beads (BangsLaboratories Inc.). Wt RecA or mutant RecA17, RecA730 or RecA17/730 were

immobilized on the bead-DNA with a free 3-end, as described in Supplementary Figure 2.Wt RecA was also immobilized on bead-DNA with a free 5-end, where the 30 mer was

biotinylated near the 3-end. After 3 washes and an incubation of 40 min at 37 C, thebeads were separated from any dissociated RecA with a magnet, resuspended in bufferand an aliquot was loaded on a protein gel. The amounts of Wt and mutant RecA on theDNA-beads were compared using two independent trials and techniques. First, a Westernblot was performed using antibodies prepared against RecA protein. We verified that the

concentrations of RecA were within the linear range (upper panel). The amounts of Wt andmutant RecA that remained on the DNA beads after 40 min were compared to a RecA

standard titration on the same blot, and relative RecA amounts were calculated. Theexperiment was repeated, aliquots were separated on a SDS protein gel and stained withImperialTM Protein Stain (Pierce). Again, the amounts of Wt and mutant RecA werecalculated by comparing the band intensities to a standard RecA titration on the same gel.The calculated amounts for each trial were averaged and are shown in the bar graph(lower panel). The error bars represent the standard error of the mean. A directmeasurement of RecA concentrations on DNA beads by spectrometry was not possiblebecause of the presence of Streptavidin on the beads.


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++RecA

+

+

3

+

+

4

/

++Pol IV

21

St

50 nt oh

St


DNA synthesis is inhibited in the presence of RecA filaments. The reactions are carried outin the presence of continuously regenerated ATP on hairpin DNA containing a 50 nttemplate overhang with Streptavidin-Biotin barriers (ST) at each end. Pol IV (5 nM) is ableto extend the hairpin (lane 1). However, the addition of RecA (4 M) to the reaction inhibitspol IV (lane 2). The presence of the sliding clamp (200 nM), loaded on the hairpin by

clamp loading complex (50 nM), strongly enhances pol IV synthesis (lane 3). Nevertheless,the reaction is inhibited on the presence of RecA despite of the presence of the clamp

(lane 4). Since Pol V does not show appreciable synthesis activity on hairpin DNA in theabsence oftrans ssDNA, it cannot be used to study RecA inhibition of DNA synthesis onthe hairpin p/t DNA. Therefore,pol IV was used to measure the effects ofbona fide RecA

filaments on DNA synthesis (see Supplementary Fig. 8).


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Sequences for template and transactivating DNA

HP 3 nt oh

5' AGA GCA GTT AGC GCA TTC AGC TCA TAC TGC TGA ATG CGC TAA CTG C 3'

trans 80mer

5' GTA TTT TCT ACG TTT GCT AAC ATA CTT CGT AAT AAG GAG TCT TAA TCA TGT TAC TGG TAA TAA GTT TTA ACG GGG

TCA GT 3'

trans 6mer

5 TAT TGT

(trans) HP 12 nt oh with biotin dT (Bio)

5' BioGA TAC AGG TGT GCA GTT AGC GCA TTC AGC BioCA TAC TGC TGA ATG CGC TAA CTG C

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