The researchers are studying the role of Mcm10 in the S-phase cell cycle checkpoint and DNA damage signaling pathways. They aim to use FRET to analyze interactions between Mcm10-YFP and Mrc1-CFP, Pol2-CFP, Dpb11-CFP, and Dpb2-CFP in yeast cells. They have optimized PCR to amplify these genes with CFP tags and are constructing yeast strains. Once complete, FRET will determine if Mcm10 interacts with these proteins during normal replication and in response to DNA damage. This could provide insight into Mcm10's role in checkpoint activation and origin licensing.

1. MCS
Integrating
Fragment
2,492 bp
Not1
Not1 Forward Primer, 32-62
Reverse Primer,
2453-2471
MCS
CFP
KAN
AMP
pDH3
4882 pb
References
K
Abstract
Introduction
Methodology
Conclusion
Cell cycle checkpoint proteins delay DNA replication to allow for repair
of damaged DNA or allow for apoptotic processes. Cancerous cells
bypass these checkpoint mechanisms. A greater understanding of
these checkpoint mechanisms could provide possible targets in anti-
cancer therapies. Minichromosome maintenance protein 10 (Mcm10)
has been previously found to be involved in DNA damage signaling with
the 9-1-1 clamp during the G1 phase of the cell cycle (1). Using yeast
two hybrid techniques, our lab has found that Mcm10 interacts with
Mrc1 and the C-terminus of Pol2 which is the catalytic subunit of
Polymerase epsilon (Pol ε). Mrc1, Pol ε, and DNA polymerase B 11
(Dpb11) are essential for cell viability and work in a complex to signal
for S phase checkpoint (2,3). The goal of our project is to observe if
Mcm10 is part of this signaling complex. To pursue this goal, we will be
using FRET to study these interactions. We will create double-tagged
strains of Mcm10-YFP with Mrc1-CFP/ Pol2-CFP/ Dpb11-CFP/ Dpb2-
CFP by homologous recombination. These strains will be sequenced to
confirm the correct integration of the fluorescent tags on the genome.
We wish to extend these studies to cells exposed to DNA damaging
conditions. We hypothesize that Mcm10 will closely interact with Pol2,
Mrc1, and Dpb11 during S phase and DNA damage, and serve as a
component of the checkpoint control pathway.
Fig. 1: Proteins at a replication fork (modified from 8)
Fig. 6: Fluorescence Resonance
Energy Transfer (FRET) Interaction
Future Directions
Acknowledgements
We would like to thank Dr. Eric Muller at the Yeast Resource Center at
University of Washington for providing the plasmids and advice.
This project was supported by the National Institute of General Medical
Sciences of the National Institutes of Health through Grant Number
8P20GM103447.
Our lab focuses on the proteins involved in the S-phase cell cycle
checkpoint and DNA replication timing, specifically Mcm10.
Mcm10, the nuclear chaperone, is essential for cell viability and has
many critical cellular roles. Mcm10 recruits Mcm2-7 helicase to the
origin and promotes DNA origin unwinding (4). Pol α, responsible for
initiating replication on both the leading and lagging strands, is
recruited to the origin of replication and stabilized by the Mcm10
interaction (5). Diubiquinated Mcm10 interacts with PCNA during late
G1 – S phase (6) and is shown to interact with the 9-1-1 clamp when
DNA is damaged by UV light or nucleotide depletion (1). All of these
interactions culminate in a protein whose mutations are found in over
41 types of cancers (7).
Fig. 7 shows the PCR optimization process with POL2, which resulted in
a specified band for gel extraction and transformation. That optimization
protocol was used with MRC1, DPB11, and DPB2 giving specified bands.
These are now prepared for gel extraction. Yeast strain DB001 (MCM10-
YFP), have been made competent and are prepared for transformation.
Department of Natural Sciences, Northeastern State University, Broken Arrow, OK 74014
Shaina Riggs, Joseph Cameron, and Brandy Fultz Faculty Mentor: Sapna Das-Bradoo
In Vivo Interactions of Mcm10 and S Phase Checkpoint Proteins Analyzed Using FRET
Results
Fig. 8: Successful digestion of pDH3 was followed by purification and
PCR amplification using the optimal protocol for PCR primers with
flanking ends for MRC1, DPB11. and DPB2.
Digested
Plasmid
Digested
Plasmid
MRC1 DPB2 DPB2 DPB2
Neg Control
DPB11 DPB11
Neg Control
DPB11
3 kb
2 kb
Yeast strains are being constructed via homologous recombination technique using
the integrating plasmid pDH3 described in Figure 3.
Fig. 3: CFP plasmid pDH3
Fig. 2: Role of Mcm10 in origin unwinding (7)
5’
5’
3’
3’
1- Alver, R., Zhang, T., Josephrajan, A., Fultz, B., Hendrix, C., Das-Bradoo, S., & Bielinsky, A. (2014).
The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA
damage. Nucleic Acids Research, 42(13), 8389-8404.
2- Osborn, A.J., Elledge S.J. (2003). Mrc1 is a replication fork component whose phosphorylation in
response to DNA replication stress activates Rad53. Genes & Development. 17, 1755-1767.
3- Araki,H., Leem,S.H., Phongdara,A. and Sugino,A. (1995). Dpb11, which interacts with DNA
polymerase II (epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a
cell cycle checkpoint. Proc. Natl. Acad. Sci. U.S.A. 92, 11791–11795.
4- Fatoba, S., Tognetti, S., Berto, M., Leo, E., Mulvey, C., Godovac-Zimmermann, J., Pommier, Y., &
Okorokov, A. (2013). Human SIRT1 regulates DNA binding and stability of the Mcm10 DNA replication
factor via deacetylation. Nucleic Acids Res., 41(7), 4065–4079.
5- Chattopadhyay, S and Bielinsky AK. (2007). Human Mcm10 regulates the catalytic subunit of DNA
polymerase-alpha and prevents DNA damage during replication. Mol Bio of the Cell. 18(10), 4085-95.
6-. Das-Bradoo, S., Ricke, R., & Bielinsky, A. (2006). Interaction between PCNA and Diubiquitinated
Mcm10 Is Essential for Cell Growth in Budding Yeast. Molecular and Cellular Biology, 26(13), 4806-
4817.
7- Thu, Y. M., & Bielinsky, A.-K. (2013). Enigmatic roles of Mcm10 in DNA replication. Trends in
Biochemical Sciences, 38(4), 184–194.
8- Sabatinos, Sarah A., et al. "Replication Fork Stalling and the Fork Protection Complex."Nature 3.9
(2010): 40.
9- Muramatsu, S., Hirai, K., Tak, Y.-S., Kamimura, Y., & Araki, H. (2010). CDK-dependent complex
formation between replication proteins Dpb11, Sld2, Pol ɛ, and GINS in budding yeast. Genes &
Development, 24(6), 602–612.
Fig. 7: Gel electrophoresis of PCR purification optimization using the
Platinum Pfx polymerase. Fig. A: standard protocol without enhancer.
Fig. B: standard protocol with enhancer and a 55o C annealing
temperature. Fig. C: lane 1 annealing temperatures were optimized to
58o C and lane 2 used 10 cycles at 58o C and 20 cycles at 64o C. Fig. D:
Preparation for gel extraction using the optimal protocol of 58o C
annealing temperatures with enhancer.
10. Fluorescence
microscopy to
confirm the presence
of the tagged
proteins.
Fig. 5: Example: DB001
Yeast Strain (MCM10-YFP)
11. Samples will be
sent for sequencing to
confirm the correct
integration of CFP on
the C-terminus of
genomic Pol2.
12. FRET analysis will allow
us quantify protein
interaction intensities in vivo
at normal concentrations
13. Cells will be synchronized
and exposed to DNA
damaging conditions during
FRET to determine if Mcm10
is part of Origin licensing or
cell cycle signaling!
Questions we aim to answer.
• Mcm10 interacts with Pol2 through its checkpoint domain and
interacts with Mrc1, a known S-phase checkpoint activation protein.
Does Mcm10 have a novel role in activation of the DNA damage
pathway on the leading strand? If so, which kinds of DNA damage
and at which checkpoints?
• Currently in the scientific field there is a disagreement if Mcm10 is
part of the pre-loading complex that helps to initiate DNA
replication.(6 & 9).
Using synchronization of cells and FRET analysis, we will determine
if Mcm10 interacts with Pol2 or Dpb11 during DNA replication
initiation.
Pol2-CFP was successfully amplified for transformation into yeast
PCR conditions were optimized to successfully amplify Mrc1,
Dpb2 and Dpb11

2. 5. Primers
designed for
POL2, MRC1,
DPB11 and
DPB2
6. PCR
optimization
using Platinum
Pfx Polymerase
7. Gel extraction
of the ~2.5 kb
integrating
vector
8. Yeast strain
DB001 (MCM10-
YFP) is made
chemically
competent
9. Transformation
of MCM10-YFP
yeast cells
1. Growth of
plasmid pDH3
in E. coli on
LB-amp
2. Miniprep
plasmid
extraction
3. Restriction
Enzyme
digestion
with Not1
4. PCR
purification
1. Growth of
plasmid pDH3
in E. coli on
LB-amp

3. MCS
Integrating
Fragment
2,492 bp
Not1
Not1 Forward Primer, 32-62
Reverse Primer, 2453-2471
MCS
Mcm10
YFP
Pol2/ Mrc1
Dpb11/Dpb2
CFP
< 10 nm
3 kb
2 kb
Expected band
POL2 MRC1
3 kb
MRC1
Expected band of
2.5 kb is absent
MRC1
Neg Control
POL2MRC1
POL2
Neg Control
POL2
2 kb
POL2
58o C
POL2
Dual Cycle
3 kb
2 kb
3 kb
2 kb
Unspecified,
Expected band
POL2 MRC1A. B. POL2
2 kb
3 kbSpecified,
Expected band
C. D.
3 kb
MRC1
Expected band of
2.5 kb is absent
MRC1
Neg Control
POL2MRC1
POL2
Neg Control
POL2
2 kb
POL2
58o C
POL2
Dual Cycle
3 kb
2 kb
3 kb
2 kb
Unspecified,
Expected band
POL2 MRC1A. B. POL2
2 kb
3 kb
~2.5 kb POL2 band
for gel purification
Specified,
Expected band
C. D.
Digested
Plasmid
Digested
Plasmid
MRC1 DPB2 DPB2 DPB2
Neg Control
DPB11 DPB11
Neg Control
DPB11
3 kb
2 kb
3 kb
MRC1
Expected band of
2.5 kb is absent
MRC1
Neg Control
POL2MRC1
POL2
Neg Control
POL2
2 kb
POL2
58o C
POL2
Dual Cycle
3 kb
2 kb
3 kb
2 kb
Unspecified,
Expected band
POL2 MRC1A. B. POL2
2 kb
3 kb
~2.5 kb POL2 band
for gel purification
Specified,
Expected band
C. D.
154880
Stop
148212
Start
Fig. 4: Yeast Chromosome XIV
Forward primer
3’ 5’
5’ 3’
Reverse primer
40
149000 154000153000152000151000150000 156000
POL2
40
ORC5
154880
Forward primer
3’ 5’
5’ 3’
40
153000152000 154000
POL2 ORC5CFP
40
KAN
157319
Total Length 2439 bp
Terminator