Download BACKGROUND CONCLUSIONS GOAL Define the protein YbfE’s role in helping

Graduate
Category: Physical and Life Sciences
Degree Level: Masters
E. coli Genes Versus % Survival at T90 Following Exposure to:
Abstract ID# 1288
Unknown
Function
yebG
Caitlin Kramer, Mark Muenter, Becky Leifer, Meghan Travers, Penny Beuning
Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
sulA
BACKGROUND
④
domains in RHH proteins – the N-terminal 74 amino acids in
YbfE might serve such a role.
• Hydropathy and disorder plots predict a disordered,
hydrophilic region from residues 42-75 connecting
an ordered N-terminus and the RHH domain.
• Circular dichroism suggests the predominant
secondary structure of YbfE is α-helical, which
supports the homology model and secondary structure
predictions.
③ The RecA-ssDNA filament facilitates the autocatalytic cleavage of the
LexA repressor, which unblocks transcription of specific SOS genes.
③
recN
④ SOS proteins, including YbfE, are expressed.
⑤
②
⑤ The SOS proteins function in a variety of ways to repair, bypass, and
adapt to damage –ultimately restoring normal replication activities.
Strains lacking the SOS protein YbfE are more sensitive to alkylation damage.
ABSTRACT
Chloroacetaldehyde
Styrene Oxide
nfo
The transcription of DNA
response genes in
prokaryotes is largely regulated
by
LexA
and
controlled
F
through the SOS response. uOver 50 genes have been
n regulon by their upstream
identified as part of the LexA
c
LexA binding sequences. Prior
work by our lab has shown
t
i
that loss of the uncharacterized
LexA-regulated gene ybfE
o
is associated with poor survival
in E. coli exposed to
n
s elucidate a mechanism, the
alkylating agents. In order to
structure and function of the ybfE gene product were
examined. A homology model was built that indicates
that YbfE is a DNA-binding protein that contains a Cterminal ribbon-helix-helix motif and a domain of
unknown function at its N-terminus. The in vivo
transcription start site of YbfE is not known, therefore
0
10 of the 20
two potential open reading frames downstream
SOS operator were examined in vitro for sequencespecific DNA binding. The first open reading frame
contains an additional 23 amino acids at the N-terminus.
Both open reading frames were cloned, expressed, and
purified. Electrophoretic mobility shift assays (EMSAs)
were performed using the YbfE protein and DNA
sequences selected to test binding specificity. Our
observations support that YbfE binds DNA nonspecifically
in vitro at concentrations above 500 nM. Characterization
of sequence specific DNA binding and protein-protein
interactions is ongoing. In vivo phenotypic experiments
show that overexpression of the ybfE gene is lethal. Sitedirected mutagenesis is being used to identify functional
residues associated with the overexpression phenotype.
recA
umuDC
umuD
Benzyl Bromide
Over 20 strains containing deletions for a variety of SOS
and DNA maintenance genes (not all data shown) were
exposed to the DNA alkylating agents chloroacetaldehyde,
styrene oxide, and benzyl bromide.
• Overexpression of YbfE is lethal to cells.
• YbfE forms dimers that can be cross-linked by
The ΔybfE strain was notably more sensitive than wildtype to all three agents.
There is no prior published research on YbfE’s structure
and function in the cell.
formaldehyde.
• EMSAs show that YbfE binds DNA.
umuC
uvrC
RESULTS
uvrA
REFERENCES
YbfE’s α-helical secondary
supported by circular dichroism
YbfE contains a DNA-binding ribbonhelix-helix motif from residues 75-109.
structure
•
dinG
?
1.20E+09
50
1.00E+09
25
alkB
ada
Circular dichroism of YbfE24-120
RHH domain
Hydrophobicity
alkylation
alkylation
DR
② Protein RecA polymerizes the single-stranded DNA past the replication
fork.
•
8.00E+08
Purified YbfE24-120
20
6.00E+08
4.00E+08
10
30
40
50
60
70
80
•
2.00E+08
0.00E+00
188
90
-2.00E+08
198
208
218
228
238
248
258
•
-4.00E+08
% Survival (T90)
DNA-binding function supported by EMSA
-6.00E+08
-8.00E+08
•
λ (nm)
-1.00E+09
[YbfE]:
•
Overexpression of YbfE24-120 and YbfE1-120
is lethal at 30, 37, and 42 ˚C
Bound
DNA
Romero, P., Z. Obradovic, C.R. Kissinger, J.E. Villafranca, and A.K. Dunker. Identifying
Disordered Regions in Proteins from Amino Acid Sequences. Proc. I.E.E.E. International
Conference on Neural Networks, 1997, p. 90-95. http://www.pondr.com
Kyte, J. and Doolittle, R.F. A simple method for displaying the hydropathic character of
a protein. J. Mol. Biol. 157, 105-132 (1982).
Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software
Suite. Trends Genet. 2000 Jun;16(6) 276-277.
http://www.ebi.ac.uk/Tools/seqstats/emboss_pepwindow/help/
Greenfield NJ. Using circular dichroism spectra to estimate protein secondary
structure. Nature protocols. 2006;1(6):2876-2890.
Hellman LM, Fried MG. Electrophoretic Mobility Shift Assay (EMSA) for Detecting
Protein-Nucleic Acid Interactions. Nature Protocols. 2007;2(8):1849-1861.
Krieger E, Koraimann G, Vriend G. 2002. Increasing the precision of comparative
models with YASARA NOVA—A self-parameterizing force field. Proteins 47: 393–402.
1.00E+01
Unbound
DNA
ACKNOWLEDGEMENTS
1.00E+00
0
0.2
0.4
0.6
0.8
1
1.00E-01
YbfE homology model – predicted key
residues highlighted
1.00E-02
YbfE forms cross-linked dimers in
presence of formaldehyde
Mins: 0
5
10
20
30
log % survival
REC &
Repair
①
ruvA
recE
DNA
NER; UV;
strand
DNA
exinucleas SOS; Pol SOS; Pol SOS; Pol exchange Endonuc.
e
& REC
IV
V
V
V
helicase NER; UV
ribbon-helix-helix
(RHH) DNA binding motif in the C-terminus of
the YbfE protein. There are often additional functional
• Sequence homology predicts a
① DNA damage causes a lesions that the main DNA polymerase Pol III
cannot bypass.
rnt
recJ
G
e
n
e
damage
CONCLUSIONS
The prokaryotic SOS response results in expression of specialized DNA-damage response enzymes.
mug
ssDNA
Exonuc.
a
n
d
mutM
Exonuc.
VIII
e
s
Hol.
Junct.
REC
GOAL
Define the proteinGYbfE’s role in helping
e
bacteria survive
DNA damage.
n
θ
SOS cell
DNA
DNA
Rnase T;
div.
degrades glycosylas glycosylas
inhibitor
e
e
tRNA
ybfE
SOS;
Binds
DNA
Benzyl Bromide; Styrene Oxide; and Chloroacetaldehyde
1.00E-03
1.00E-04
1.00E-05
Arg-71
1.00E-06
dimer
24NSPybf
E - 37
24NSPybf
E - 42
1.00E-07
Arg-75
Leu-78
monomer
1.00E-08
[IPTG], mM
Members of the Beuning DNA Lab – past and
present
Dr. Thomas Wales and Dr. John Engen for use of
their spectropolarimeter
Dr. Mary Jo Ondrechen and CHEM 5683 SP15
The American Cancer Society
Northeastern University
Provost Thesis Grant