WEEK 1

DAY 1: 6/6/17 
I was super nervous for my first day and I didn't know what to really expect! Once I arrived, I was greeted by Dr. MacNeill, and we went through the plan for the week. He showed me around and where everything was in the lab. My first task was to read all the healthy and safety rules on my cubicle (yes, I got a whole cubicle desk for myself.. how cool is that?! super cool I say!). Once I read everything, I was ready to head to the lab, and feel like a true scientist.

Dr. MacNeill had prepared beforehand 4 plasmids (he attempted to make 5, but for some unknown reason, the 5th plasmid just wasn't having any of it, and decided not to work). Today in the lab, I attempted to mutate each binding site found in the cdc27 (p66 in humans) subunit. Plasmid 1 had its phosphorylation sites mutated. Plasmid 2 had one of its ubiquitylation sites mutated. Plasmid 3 had its sumoylation (SUMO) site mutated. Each plasmid was transformed into cdc27/cdc27del yeast diploid cells and incubated at 32oC for about a week (to allow cells to grow and colonies to form).


DAY 2: 7/6/17
Today's task was to cut, purify and ligate DNAs (plasmid sample and insert DNA), then transform E. coli. The plasmid used as N11 plasmid, and it was digested with NotI-HB restriction enzyme. The inner DNA cdc27 (spcdc27-HBT) alleles were also digested with NotI-HB restriction enzyme. Before ligating both the plasmid sample and the insert DNA together, their solutions were run in an agarose gel, against a DNA molecular weight tag, for comparison (see figure 1). Once ligation was done, a 50ul and a 150ul solutions were placed in LB+ ampicillin plates and insulated overnight, allowing transformation to occur.
NOTES: bacteria was used for transformation because it grows quicker than yeast.

From figure 1, it can be seen that the plasmid is A and that the insert DNA is B. This can be told, not only by being organised and labelling the solutions being placed in the agarose gel, but also, because of the molecular weight of both solutions. The insert DNA is smaller than the plasmid, which means it will travel further in the agarose gel.

DAY 3: 8/6/17
Today's task was to screen colonies by colony PCR and set up culture for mini-preps. However, one of the enzymes required for colony PCR hadn't arrived yet (it was still in Germany.. so we waited). However, I still did some lab work: the plasmids prepared on DAY 2 were grown, and 8 colonies were placed into 8x tubes containing LB Broth and ampicillin, and incubated in a shaker overnight to form bacterial culture.

NOTES: two primers will bind to the transformed plasmid, however, a signal will only be transmitted if both primers are pointed to each other (correct orientation). If it occurs that more than one insert DNA is present in the plasmid, then the shortest sequence will be amplified (correct orientation). The longer sequences can happen to be amplified, but they will mostly likely serve as a template for the amplification of the shortest sequence. Furthermore, the longer sequences are hard to be identified in an agarose gel.

DAY 4: 9/6/17
Today, the primers arrived, so it was possible to carry on colony PCR using the bacterial culture prepared on DAY 3. All the 8 PCR solutions were allowed to undergo 25 cycles in a PCR machine (took ~1 hour 15 mins), then solutions were run in an agarose gel against a DNA MW solution, for comparison. Solutions were placed in the gel in the order: DNA MW, 1, 2, 3, 4, 5, 6, 7, 8 (see figure 2 for results).

From figure 2, it can be seen that plasmid 3 and 7 have the strongest signal, suggesting that those two plasmids underwent transformation and ended up with the right sequence at the correct orientations. So, due to the results from figure 2, it was decided to prepare/transform plasmids 3 and 7 into cdc27/cdc27del diploid following the same protocol as in DAY 1.

Introduction

This summer, I have secured an internship for the Summer of 2017 in the School of Biology at the University of St Andrews, under the supervision of Dr Stuart MacNeill. This internship opportunity is supper exciting because I will obtain hands-on experience of state-of-the-art laboratory techniques! Also Dr MacNeill has  been successful in his career. He has contributed with ~ 75 publications and is a SULSA (Scottish Universities Life Sciences Alliance) Reader in Translational Biology. Dr. MacNeill’s research is focused on chromosomal DNA replication and genome stability, and I'm really hoping to learn as much as possible from him. 

My internship will focus on chromosomal DNA replication, more specifically, in cdc27 subunit of DNA polymerase. I will try and keep you guys updated on what is happening inside the lab! Also If you're interested in extra readings, there is a few papers listed in the references section. And if you want learn a little bit more about my internship aims and why what I'm doing is relevant, please read the background information section, shown below. 

Background Information

Successful chromosomal DNA replication is essential for maintaining genome integrity in all forms of cellular life. Defective DNA replication can have a wide variety of effects on genome structure and information content, such as sequence deletion, insertion and duplication, point mutation and chromosome fusion. In mammals, defects in replication can lead to developmental defects, growth impairment and tumour formation.
The proposed work centres on two family B DNA polymerase enzymes with distinct roles in eukaryotic chromosome replication: DNA polymerase δ (Pol δ) and DNA polymerase ζ (Pol ζ). At the replication fork, Pol δ is essential for lagging strand DNA synthesis and may play a role in leading strand synthesis also (1). Pol δ also acts in a variety of DNA repair pathways and several recent studies have shown that mutations in human Pol δ bring about tumour formation. Pol ζ is translesion synthesis (TLS) polymerase (2). This enzyme replicates through sites of damaged DNA but does so with low fidelity. Pol ζ activity therefore contributes to cell survival following DNA damage, by ensuring that the chromosomes are fully replicated, but at the expense of introducing mutagenic changes into the DNA. Gaining a complete picture of the structure, function and regulation of these enzymes is crucial to fully understanding their cellular roles and the broader contributions that they make to maintaining genome integrity.
At the molecular level, human Pol δ comprises four subunits: PolD1, PolD2, PolD3 and PolD4. PolD1 (also known as p125) is the catalytic subunit, possessing polymerase and proofreading exonuclease activities, whereas PolD2 (p50), PolD3 (p66) and PolD4 (p12) are accessory subunits whose contributions to Pol δ activity remain largely unknown (3). It was recently shown that PolD2 and PolD3 are also subunits of human Pol ζ, which is a heterotetramer of PolZ1 (catalytic subunit Rev3), PolZ2 (Rev7), PolD2 (p50) and PolD3 (p66) (2).
Fission yeast is an excellent model for investigating the function of these enzymes. Like its human counterpart, fission yeast Pol δ is heterotetrameric, comprising orthologues of PolD1 (Pol3), PolD2 (Cdc1), PolD3 (Cdc27) and PolD4 (Cdm1) (4). This is not the case for the more widely studied budding yeast Pol δ enzyme, which lacks PolD4. Fission yeast Pol ζ has not been characterised biochemically but is likely composed of Rev3, Rev7, Cdc1 and Cdc27.
The proposed project focuses on Pol δ/Pol ζ shared subunit Cdc27, the fission yeast orthologue of human PolD3 (p66), and its potential regulation by post-translation modification (PTM). Cdc27 is a 372 amino acid protein. The first 160 amino acids bind to Cdc1, the fission yeast PolD2 protein, while interaction sites for Pol α–primase and the sliding clamp processivity factor PCNA are found in the C- terminal region of protein (spanning residues 293-332 and 362-369, respectively) (5,6). By comparison with human PolD3 (p66) (7), the N-terminal region likely forms a winged helix-turn-helix (wHTH) domain. In contrast, the C-terminal region may be largely unstructured.
Recent proteome-wide analysis has identified two ubiquitylation (Lys185, Lys187) (8), one sumoylation (Lys317) (9) and five phosphorylation (Ser160, Thr161, Ser163, Ser167, Thr187) (10,11) sites within the C-terminal domain of Cdc27 protein. The sumoylation site maps within the minimal Pol α–primase interaction motif mentioned above (6), suggesting that sumoylation may function to modulate this interaction. None of these PTMs is essential for Pol δ function, as we have previously shown that cells expressing a truncated protein lacking amino acids 161-372 are viable (12). Aside from this, the functions of the PTMs have not been investigated.

Objectives
The overarching aim of the proposed work is to gain insight into the potential roles of ubiquitylation and sumoylation in modulating PolD3 function. Using fission yeast as a model system, the student will use a combination of biochemical and molecular genetic methods to investigate the role(s) of these PTMs. Through this work, we hope to provide evidence that the identified modifications play a role in modulating the properties of Cdc27, thereby opening up new avenues of research into the regulation of Pol δ and Pol ζ activity in yeast and ultimately, human cells. 

References
1. Lujan, S.A. et al. (2016) Trends Cell Biol, 26, 640-654.
2. Makarova, A.V. and Burgers, P.M. (2015) DNA Repair (Amst), 29, 47-55.

3. Tahirov, T.H. (2012) Subcell Biochem, 62, 217-236.

4.   Zuo, S. et al. (1997) Proc Natl Acad Sci U S A, 94, 11244-11249.
5.  Gray, F.C. et al. (2004) BMC Mol Biol, 5, 21.
6.  Reynolds, N. et al. (2000) EMBO J, 19, 1108-1118.

7. Baranovskiy, A.G. et al. (2008) Cell Cycle, 7, 3026-3036.
8. Beckley, J.R. et al. (2015) Mol Cell Proteomics, 14, 3132-3141.
9. Kohler, J.B. et al. (2015) Nat Commun, 6, 8827.
10. Koch, A. et al. (2011) Sci Signal, 4, rs6.

11. Kettenbach, A.N. et al. (2015) Mol Cell Proteomics, 14, 1275-1287.

12. Tanaka, H. et al. (2004) Nucleic Acids Res, 32, 6367-6377.