Supplementary Information for - Nature Research · 2013-10-04 · calculated (est2 n = 48, PD 30; est2 hpr1 n = 39; PD 26). a b Relative cell density est2 rad52 est2 rad52 hpr1 Average

Supplementary Information

for

Telomeric RNA-DNA hybrids affect telomere length dynamics and senescence

Bettina Balk1,2, André Maicher1,2, Martina Dees1, Julia Klermund1, Sarah Luke-

Glaser1, Katharina Bender1 and Brian Luke1,3

1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-

ZMBH Alliance, Heidelberg, Germany

2These authors contributed equally

List of Supplementary Information

Supplementary Figures (5)

Supplementary Tables (3)

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662

Supplementary Figure 1. Telomeric RNA-DNA hybrids are regulated by

RNase H enzymes

(a) RNA-DNA hybrids exist at telomeres and accumulate in RNase H mutants. The

identical ChIP as described in Fig. 1a, however quantified showing % input on the Y-

axis. Values are represented as % input of telomeric DNA recovered and are the

means of seven (+ab) and five (-ab) biological replicates, error bars depict ± s.e.m.

P values were derived from two-tailed Student’s t-tests (NS = not significant). (b) The

RNA-DNA hybrid signal is RNase H sensitive. Following overnight

immunoprecipitation with the S9.6 antibodies, one wild type sample was treated with

recombinant RNase H before the washing steps (see online methods). (c) TERRA

levels are not increased in rnh1 rnh201 mutants. qRT-PCR was performed for the

6Y’, 1L and 15L telomeres using the indicated mutants (at approx. PD 15 following

tetrad dissection). sir2 cells served as positive control where TERRA is upregulated.

Average values were derived from 3 biological replicates where error bars depict ±

s.e.m.

ba

c

WT

rnh1 rnh201

0

0.2

0.4

0.6

0.8

1.0

ab

RNase H

6Y´15L rDNA

+ +

+

1L

0

0.05

0.10

0.60

0.80

1.00

P = 0.46

ab + + +

6Y´15L rDNA

+

1L

NS

P = 0.61

P = 0.001

***

P = 0.03

*P = 0.003

**

NS

P = 0.09

**P =0.005

NS

P = 0.02

*

% I

np

ut

% I

np

ut

Fo

ld e

nrich

me

nt

co

mp

are

d t

o W

T

0

100

200

300

1

2

3

4

5

1L 15L

WT

rnh1 rnh201est2est2 rnh1 rnh201sir2

– – – –

–

– –

+ +

+

–

– –

+ +

+

–

– –

+ +

+

–

– –


Supplementary Figure 2. RNA-DNA hybrids accumulation promotes

homologous recombination

(a) Raw data from Fig. 1b. The individual pairs within a graph were derived from the

same tetrad for direct comparisons. (b) The second and third biological replicates

from Fig. 2a. (c) Premature survivor formation does not account for the increased rate

of HR in rnh1 rnh201 cells. Genomic DNA derived from tetrad 2 of the curve shown

in Fig. 1b was digested with Xho1. TRFs of Y´ telomeres can be seen between 1164

and 992 bps. Recombination rates for Fig. 2a were determined at around PD 35, when

type II survivors had clearly not yet formed.

Day 1 3 5 7 9 11 1 3 5 7 9 11

est2 est2 rnh1 rnh201

19531882

15151482

1164

992

710

2799

3639

4899

bp

PD 9 27 43 55 73 92 9 26 44 59 71 85

Type IIsurvivors

Type IIsurvivors

8

6

4

2

0

est2 est2 rnh1 rnh201 est2 est2 rnh1 rnh201

Cel

l den

sity 8

6

4

2

0

8

6

4

2

0150100500 150100500

PDs PDs

a

b

Tetrad 2

Undiverged region Diverged region

050

100150200250300350

Telomeres from est2 rnh1 rnh201 tetrad 2 – PD 36 Telomeres from est2 tetrad 2 – PD 36

11 % Divergence 18 % Divergence

Tetrad 1 Tetrad 2

Tetrad 3 Tetrad 4

Tetrad 5 Tetrad 6

cTe

lom

ere

leng

th (

bp)

12% Divergence 19% Divergence

Telomeres from est2 tetrad 3 – PD 36 Telomeres from est2 rnh1 rnh201 tetrad 3 – PD 36

est2 est2 rnh1 rnh201

050

100150200250300350

Telo

mer

e le

ngth

(bp

)


Supplementary Figure 3. Rnh1 and Rnh201 act redundantly to remove telomeric

RNA-DNA hybrids.

(a) The increased rate of senescence shown in Fig. 2c only occurs in absence of

telomerase. Senescence curves were performed as previously described (Fig. 1b). The

loss of RNase H activity does not result in decreased cell viability in rad52 cells, but

accelerates senescence only when both EST2 and RAD52 are co-deleted. Curve

averages were derived from 6 biological replicates for each genotype depicted, error

bars depict ± s.e.m. est2 rad52 and est2 rad52 rnh1 rnh201 curves are taken form

Fig. 2c. (b, c) Senescence curves in an est2 rad52 background were performed with

only one of the RNase H genes deleted (rnh1 or rnh201, respectively). Rates of

senescence were not affected upon deletion of one RNase H gene only. Curves

represent the means of 6 biological replicates ± s.e.m. (d) Raw data from Fig. 2c.

Genotypes are indicated.

0 10 20 30 40 50

0

50

100

150

est2 rad52est2 rad52 rnh1 rnh201 rad52 rnh1 rnh201

rad52R

ela

tive

ce

ll d

en

sity

Average PDs

est2 rad52

est2 rad52 rnh1

Re

lative

ce

ll d

en

sity

est2 rad52est2 rad52 rnh201

Average PDs

Re

lative

ce

ll d

en

sity

0 10 20 30 400

50

100

150

0 10 20 30 40 500

50

100

150

Average PDs

ba

c

PDs

0 10 20 30 40 50

6

4

2

0

Ce

ll d

en

sity (

OD

600)

est2 rad52 1

est2 rad52 rnh1 rnh201 1est2 rad52 rnh1 rnh201 exo1 1est2 rad52 2


est2 rad52 rnh1 rnh201 3est2 rad52 rnh1 rnh201 exo1 3

est2 rad52 6


est2 rad52 4



d


Supplementary Figure 4. The THO mutant hpr1 shows increased telomeric

RNA-DNA hybrids and increased rates of telomeric recombination.

(a) hpr1 shows increased telomeric RNA-DNA hybrids. ChIP was carried out as

described in Fig. 1a. The values are represented as % input of telomeric DNA

recovered relative to wild type (set to 1) and are the means of 5 biological replicates,

error bars depict ± s.e.m. P values were derived from a two-tailed one-sample

Student’s t-test. (b) Deletion of HPR1 accelerates senescence in HR deficient

telomerase mutants. In the presence of RAD52 the viability loss was alleviated,

suggesting HR-mediated compensation. Liquid senescence assays were performed on

the indicated strains. Curves represent the average value for 6 biological

replicates ± s.e.m. (c) hpr1 shows increased rates of telomeric recombination. 1L

Telomere-PCR was performed on genomic DNA extracted from the two indicated

clones, which were derived form the same tetrad. 1L Telomere-PCR products were

cloned and sequenced as described in Fig. 2a and % of diverged telomeres was

calculated (est2 n = 48, PD 30; est2 hpr1 n = 39; PD 26).

a b

Rel

ativ

e ce

ll de

nsity

est2 rad52est2 rad52 hpr1

Average PDs

est2 est2 hpr1

c

Div

erge

d te

lom

eres

(%)

0

5

10

15

20

est2 est2 hpr1

0.0

0.5

1.0

1.5

2.0P = 0.05

*

6Y´15L rDNA

Rel

ativ

e to

WT

(% In

put)

NSP = 0.06 P = 0.17

NS

WT

hpr1

0 20 40 60 80 1000

50

100

150


Supplementary Figure 5. Overexpression of RNH1 increases senescence in

recombination competent cells.

(a) Raw data for Fig. 5a for the indicated genotypes. (b) Raw data for Fig. 5e for the

indicated genotypes.

est2 rad52 + RNH1 3est2 rad52 + VC 3

Cell d

ensit

y (OD

600) 6

4

2

00 10 20 30 40

est2 rad52 + VC 1est2 rad52 + RNH1 1

6

4

2

00 10 20 30 40


6

4

2

0 10 20 30 40

6

4

2

0 10 20 30 40


Cell d

ensit

y (OD

600) 6

4

2

0 10 20 30 40


6

4

2

0 10 20 30 40


0

0 0 0

PD

6

4

2

0 10 20 30 40


0

PD

6

4

2

0 10 20 30 40


0

PD

Cell d

ensit

y (OD

600)

6

4

2

0 10 20 30 40


0

PD

0 20 40 60 80 1000

2

4

6

8 est2 +VC 1est2 +RNH1 1

Cell d

ensit

y (OD

600) est2 +VC 2

est2 +RNH1 2

PD

20 40 60 80 1000

2

4

6

8

PD

20 40 60 80 1000

2

4

6

8

Cell d

ensit

y (OD

600)

PD

20 40 60 80 1000

2

4

6

8

PD

20 40 60 80 1000

2

4

6

8

Cell d

ensit

y (OD

600)

PD

20 40 60 80 1000

2

4

6

8

est2 +VC 3est2 +RNH1 3




a

b


Supplementary Table 1. Yeast strains used in this study

Yeast strains used in this study were derived from BY4741 background (his3-1, leu2-

0, ura3-0, met15-0)

Code Name Genotype

YBL7 wild type MATa

YBL588 rnh1 rnh201 MATa rnh1::KAN rnh2::NAT

YAM76 hpr1 MATa hpr1::KAN

YBB 236 est2 rnh1 rnh201 MATa/MATalpha EST2/est2::HYG RNH1/rnh1::KAN

RNH201/rnh201::NAT

YBB 261 est2 rad52 +VC MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::KAN +

pRS426 GPD

YBB 261 est2 rad52 +RNH1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::KAN +

pRS426 GPD RNH1

YAM184 est2 rad52 rnh1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT

RNH1/rnh1::KAN

YAM186 est2 rad52 rnh201 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT

RNH201/rnh201::HYG

YBB238 est2 rad52 rnh1 rnh201 exoI MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT

RNH1/rnh1::KAN RNH201/rnh201::HYG EXOI/exoI::URA3

YAM188

YSLG432

est2 rad52 rnh1 rnh201

7L*

MATa/ MATalpha EST2/est2::HIS3 RAD52/rad52::NAT

RNH1/rnh1::KAN RNH201/rnh201::HYG

7L::URA3

YAM156 est2 rad52 hpr1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT

HPR1/hpr1::KAN

Supplementary Table 2. Plasmids Code Name Genotype

pBL189 VC pRS426 GPD

pBB39 RNH1 pRS426 GPD RNH1


Supplementary Table 3. Oligos Code Name Genotype Experiment

oBL358 1L GCGGTACCAGGGTTAGATTAGGGCTG Telomere-PCR

oBL360 6R AAATGAGGACTGGGTCATGG Telomere-PCR

oBL361 6Y` TTAGGGCTATGTAGAAGTGCTG Telomere-PCR

oAM26 7L CGGATCCCAGAGTAGAGGTAG Telomere-PCR

oBL359 oligo-dG CGGGATCCGGGGGGGGGGGGGGGGGG Telomere-PCR

oBL295 1L-fwd CGGTGGGTGAGTGGTAGTAAGTAGA ChIP

oBL296 1L-rev ACCCTGTCCCATTCAACCATAC ChIP

oLK49 6Y’-fwd GGCTTGGAGGAGACGTACATG ChIP

oLK50 6Y’-rev CTCGCTGTCACTCCTTACCCG ChIP

oLK57 15L-fwd GGGTAACGAGTGGGGAGGTAA ChIP

oLK58 15L-rev CAACACTACCCTAATCTAACCCTGT ChIP

oBL292 actin-fwd CCCAGGTATTGCCGAAAGAATGC ChIP

oBL293 actin-rev TTTGTTGGAAGGTAGTCAAAGAAGCC ChIP

oAM47 rDNA-fwd TCCAATTGTTCCTCGTTAAG ChIP

oAM48 rDNA-rev ATTCAGGGAGGTAGTGACAA ChIP


Documents

Supplementary Information for - Nature Research · 2013-10-04 · calculated (est2 n = 48, PD 30; est2 hpr1 n = 39; PD 26). a b Relative cell density est2 rad52 est2 rad52 hpr1 Average