Brannon et al. 10.1073/pnas.0407818102.

Supporting Information

Files in this Data Supplement:

Supporting Table 1
Supporting Table 2
Supporting Figure 7
Supporting Table 3
Supporting Table 4




Fig 7. YMB1670 transformants containing p416CYC1E2 and derived plasmids expressing mutated E2 proteins were labeled with 35S-methionine, and extracted proteins were immunoprecipitated with E2-specific antibody B201. Precipitated proteins were separated by SDS/PAGE. The position of the E2 proteins is indicated by the arrow. Although A1 and C9 proteins are at reduced levels, they are able to support plasmid maintenance.





 

Table 1. Plasmid genes, elements, and markers

 

Plasmids

Markers

Source

Reporter

    

YRp7

ARS1, TRP1

Ref. 1

YRp7-LCR

ARS1, TRP1, LCR

This study

pRS314

ARSH4, CEN6, TRP1

Ref. 2

pRS304ARS-ADE3

ARSH4, TRP1, ADE3

This study

pRS304ARS-E2RE1-ADE3

ARSH4, TRP1, ADE3, E2RE1

This study

pRS304ARS-LCR-ADE3

ARSH4, TRP1, ADE3, LCR

This study

pRS314-ADE3

ARSH4, CEN6, TRP1, ADE3

This study

E2 expression

   

pRB16 vector

ARSH4, CEN6, HIS3

Ref. 3

pRB16-E2

ARSH4, CEN6, HIS3, PADHcE2

This study

p416CYC1 vector

ARSH4, CEN6, URA3

Ref. 4

p416CYC1-E2

ARSH4, CEN6, URA3, PCYC1E2

This study

Cellular genes

    

pADNS vector

2micron, LEU2,

Ref. 5

pADNS-Brd4

2micron, LEU2, PADHBrd4

This study

pADNS-Brd2

2micron, LEU2, PADHBrd2

This study

pADNSpl-Bdf1

2micron, LEU2, PADHBdf1

This study

pADNSpl-Bdf1rcMtail

2micron, LEU2, PADHBrd4

This study

pR425/PGK vector

2micron, LEU2

Ref. 6

p425PGK-hEBP2

2micron, LEU2, PPGK hEBP2

Ref. 6

1. Stinchcomb, D. T., Struhl, K. & Davis, R. W. (1979) Nature 282, 39-43.

2. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27.

3. Brachmann, R. K., Vidal, M. & Boeke, J. D. (1996) Proc. Natl. Acad. Sci. USA 93, 4091-4095.

4. Mumberg, D., Muller, R. & Funk, M. (1995) Gene 156, 119-122.

5. Colicelli, J., Birchmeier, C., Michaeli, T., O’Neill, K., Riggs, M. & Wigler, M. (1989) Proc. Natl. Acad. Sci. USA 86, 3599-3603.

6. Kapoor, P., Shire, K. & Frappier, L. (2001) EMBO J. 20, 222-230.





Table 2. Properties of mutated E2 proteins

E2 protein

Mutation

Chromosome binding, percent mitotic cells

BRD4

binding

in vitro, percent WT

Transctivation,

percent WT

DNA replica-

tion

DNA binding

37°C

34°C

37°C

34°C

E2-TA

None

85

100

100

+++

+++

+++

+

A1

E2A, E6A, E13A, E20A

98

ND

79

+++

+++

+/-

+

C9

Q12N, R68Q

0

36

41

+/-

+

-

+

F3

R37A, I73A

0

0

3

+

+

+++

+

K342

R342K

+++

ND

ND

-

-

-

-

E2-TR

None

0

0

0

-

-

-

+

Transcriptional activation and DNA replication activity. +++, >60% activity; ++, >30-60% activity; +, >10-30% activity; ± , ≤10% activity. Data is from refs. 1 and 2 and McPhillips, M. G., Ozato, K., and A.A.M., unpublished data.

1. Baxter, M. K., McPhillips, M. G., Ozato, K. & McBride, A. A. (2005) J. Virol., in press.

2. Baxter, M. K. & McBride, A. A. (2005) Virology 332, 78-88.





Table 3. Plasmid retention activity of mutated E2 proteins

Percent retention of reporter plasmid

ARS

E2RE1/ARS

LCR/ARS

CEN/ARS

Vector

8.4

9.8

8.2

87.0

E2

2.2

30.4

34.6

90.6

E2 K342

5.6

6.5

4.5

111.1

E2-TR

5.6

6.0

5.6

47.7

E2 F3

6.2

8.7

9.4

94.3

E2 C9

4.5

18.4

18.8

76.6

E2 A1

5.3

18.1

30.2

106.6

Representative liquid culture assay of YMB1670 transformants with pADNS-Brd4, individual reporter plasmids (see Fig. 1B), and p416CYC1 plasmids expressing WT or mutated E2 proteins, as indicated. Percent plasmid retention was calculated by dividing the number of colonies on the selective plates by the number of colonies on the nonselective plates.





Table 4. Bdf1-BRD4 fusion protein supports E2-mediated plasmid maintenance

Percent retention of reporter plasmid

ARS

E2RE1/ARS

LCR/ARS

CEN/ARS

Vector

6.2

4.7

5.3

81.7

Brd4

2.3

31.5

41.5

93.9

BDF1Mtail

1.9

5.0

11.3

49.3

Representative liquid culture assay of YMB1670 transformants with p416CYC-E2, individual reporter plasmids (see Fig. 1B), and pADNS-Brd4, pADNSpl, and pADNSpl-Bdf1rcMtail expression plasmids. Percent plasmid retention was calculated by dividing the number of colonies on the selective plates by the number of colonies on the nonselective plates.