Download Career Development Plan-Year 1 Analysis of DNA looping by Type

Career Development Plan-Year 1
Name of Fellow: Christian Pernstich
Department: Biochemistry, University of Bristol, UK
Name of Supervisor: Professor S. E. Halford
Name of International Co-assessor for the RTN: Dr V. Siksnys
Name of Co-assessor for the University of Bristol: Dr. D. Stephens
Start Date: July 1st, 2006
BRIEF OVERVIEW OF RESEARCH PROJECT AND MAJOR ACCOMPLISHMENTS EXPECTED
Analysis of DNA looping by Type II Restriction Enzymes
Restriction endonucleases occur ubiquitously among prokaryotic organisms. Their principal
biological function is the protection of the host genome against foreign DNA, in particular
bacteriophage DNA. By definition, restriction endonucleases are part of the restrictionmodification (RM) system, which comprises an endonuclease and a methylase activity. Four
types of RM systems have been found and are classified according to their subunit
composition, cofactor requirement and mode of action.
This project will focus on Type II restriction endonucleases. The main criterion for
classifying a restriction endonuclease as a Type II enzyme is that it cleaves DNA specifically
within or close to their recognition site and they do not require ATP hydrolysis for nucleolytic
activity.
Recently, it has become clear that Type II restriction endonucleases do not conform to
this narrow definition, since they show a wide range of recognition sites and reaction
mechanisms, making it necessary to divide further into subfamilies according to their cleavage
patterns and mode of action. For example, the Type IIs restriction endonucleases, such as FokI,
recognise asymmetric sequences and cleave at defined distances away from these sequences.
The aim of the project is to investigate the protein-DNA complexes of Type II
restriction enzymes that interact with two separate sites in DNA, especially conformational
changes in the protein, as well as the process of DNA looping itself. The enzymes that will be
examined here include FokI, which exists in solution as a monomer but in which the catalytic
domain is out of position to interact with DNA. It also has to dimerise in order to cut both
strands of the DNA, but the dimer forms more readily on DNA with two sites than on DNA
with one site. These processes will be examined by fluorescence methods including FRET and
by single molecule methods using optical traps.
LONG-TERM CAREER OBJECTIVES (over 5 years):
1. Goals:
• To obtain a higher degree (Ph.D.) through a research-based programme and the
submission of a research dissertation
• To gain experience in modern biochemistry and molecular biology techniques
for the study of molecular genetics
• To obtain a further post in the sciences
2. What further research activity or other training is needed to attain these goals?
• To plan and undertake a three year programme of laboratory-based research,
studying reaction mechanisms of Type II restriction enzymes
• To produce publications, both in international journals and as part of a written
thesis.
• To collaborate with other research groups (within Bristol, within the EU Marie
Curie consortium and with other international groups)
• To participate in conferences and workshops
• To participate in training courses within the University of Bristol and beyond,
wherever appropriate
• To maintain a current CV so that future employment goals can be planned for
and met
SHORT-TERM OBJECTIVES (1-2 years):
1. Research results
Research goals (overall): How do selected Type II enzymes loop and cleave DNA in
detail?
Research goals (year 1):
(i)
To produce a set of FokI mutants by exchanging either the
cysteine in the DNA recognition domain, or the cysteine in
the DNA cleavage domain, with serine residues. To test for
changes in the activities of these mutants
(ii)
To modify the mutant proteins at their individual cys residues
with suitable fluorophores, and to test the modified proteins
for activity.
(iii) To use the fluorescently-labelled proteins in FRET and
anisotropy experiments in order to measure the proteinprotein and domain-domain interactions of FokI.
(iv)
To construct suitable DNA substrates with fluorescent labels,
for protein-DNA FRET, and for single molecule experiments
with optical traps.
Research goals (years 2 & 3)
These will be become clearer based on the results of the experiments in the first year.
Anticipated conference, workshop attendance, courses, and /or seminar presentations:
Suitable Conferences
Annual Meetings of the EU “DNA-enzymes” consortium (Bristol 2006, Vilnius 2007, Berlin
2008, Giessen 2009)
NACON VIII , Nucleic Acid Recognition, (Sheffield, Easter 2007)
FASEB meeting of “Nucleic Acid Enzymes” (Vermont, Summer 2008)
Other conferences on “Enzymes acting on DNA”, to be identified when advertised
Suitable Workshops
Workshops of the EU “DNA-enzymes” consortium (Odense 2006, Warsaw 2007, York 2008,
Giessen 2009)
Masters Course in Biophysikalischen Chemie (Dusseldorf, 2006)
To be identified when advertised
Courses
Suitable courses run by University of Bristol Staff Development
(http://www.iser.bris.ac.uk/cgi-bin/sdev/courselist.pl). Possibilities include:
Planning and managing your PhD (SD1PHD)
Writing successful grant proposals (SD1WGP) (in year 3)
Completing your PhD - when the going gets tough (SD3PHD) (in year 3)
Presentation skills (SD1MAP)
To be identified when advertised
Seminar Presentations
Annual Meetings of the EU “DNA-enzymes” consortium (Bristol 2006, Vilnius 2007, Berlin
2008, Giessen 2009)
Ph.D. presentations to the Department of Biochemistry, University of Bristol (years 1 & 3)
Internal meetings of the DNA-Protein Interactions Unit, Department of Biochemistry,
University of Bristol
2. Research Skills and techniques:
o Experimental Planning, e.g., plasmid design; designing and ordering
oligonucleotides; anticipated use of chemicals, enzymes, etc.; risk assessment
where appropriate.
o Experimental Procedure, e.g., technical skills in biochemistry and molecular
biology appropriate to the study of DNA-protein interactions; safe working in a
lab environment; efficient use of lab notebooks, etc.
o Experimental Analysis, e.g., self-critical thinking; computer programmes for
presentation, analysis and modelling; record keeping, etc.
o Experimental Presentation, e.g., weekly lab meetings; monthly Supergroups;
yearly departmental seminars; poster preparation and presentation; paper
writing, submission and publication.
3. Research management:
o Within the Halford Group, Bristol – planning experiments and resources,
achieving deadlines, time management, managing the Ph.D., communications
with group members
o Within the DNA-Protein Interactions Unit, Bristol – maintaining lab space
(lab chores), maintenance and stocktaking of communal equipment and
materials, communications with members of other groups in the unit
o Within the DNA-Enzymes consortium – identifying suitable collaborative
partners, organising visits, communications with members of the consortium
o Wider research community – communications with other members of the
University of Bristol (e.g., at the departmental retreat, Sept 2006) and the wider
research community (e.g., at research conferences, workshops, etc).
Communication skills:
Personal presentation skills, poster presentations, skills in report writing and preparing
academic papers and books.
Training provided in efficient use of lab notebooks and computer files/folders
Weekly lab meetings and regular meetings with supervisor (at least fortnightly)
Yearly Ph.D. reports required which are assessed by an internal co-assessor
Opportunity to prepare draft of any papers arising from project
Shared research lab (6 groups in D40) gives opportunity to develop interpersonal skills
To be able to defend research outcomes at seminars, conferences, etc.
Weekly lab meetings and monthly supergroups allow presentation and defence of research
Biochemistry department talks in years 1 & 3 allow presentation and defence of research
Poster presentation and defence REQUIRED for attendance at any conference
Opportunities for other research presentations to be identified
Contribute to promote public understanding of one’s own field
Opportunity to participate in “Schools Week” and “Science Alive”
Other opportunities to be identified – e.g., Essay competitions (in year 3)
4. Other professional training (course work, teaching activity):
Course work
Mandatory courses (inc. safety courses) as part of the graduate school of the faculty of medical
and veterinary sciences (see point 1, above)
Teaching/Supervision
Opportunities for assistance with teaching of undergraduates in the Biochemistry Department
(e.g., as an unpaid lab practical demonstrator) – years 1-3
Opportunities for supervision of third year undergraduates from Biochemistry Department
during their 8 week lab placement – years 2 or 3
Opportunities for mentoring of new members of the lab – years 1-3
5. Anticipated networking opportunities
Internal – Weekly lab meetings, monthly supergroups, weekly departmental seminars, biennial
departmental retreats
External – DNA-Enzymes annual meetings and workshops, other conferences (poster
presentation is important to achieve this), other courses and workshops (to be identified)
6. Other activities (community, etc) with professional relevance:
None anticipated at present
10th October, 2006
Date & Signature of fellow:
10th October, 2006
Date & Signature of supervisor