Dr Ian Grainge

Biography

I am interested in all aspects of how bacteria pass on their genetic information, from DNA replication to chromosome segregation and accurate cell division.

My current research focuses on two main topics. Firstly what happens when the process of DNA replication runs into a blockage and stops- how can the cell recover to restart the vital process of copying its DNA? A large number of homologous recombination proteins have been implicated in the processing of collapsed DNA replication forks and their roles, and the pathways used, will be investigated in living cells.

Secondly, is the study of the FtsK protein, which co-ordinates the processes of cell division, chromosome unlinking and chromosome segregation in bacteria. Each of these processes has to be completed in a timely manner to allow the cell to divide to produce offspring with a full genetic content. The DNA translocase protein, FtsK, is a key protein in each of these processes, and could additionally act as a cell division checkpoint.

Post-doctoral work:

Department of Biochemistry, University of Oxford (2005-2009)

Cancer Research UK (2000-2004)

University of Texas at Austin (1997-2000)

PhD:

University of Oxford, UK (1994-1997)

Qualifications

• PhD, University of Oxford - UK, 1999
• Master of Arts, University of Cambridge - UK, 1998
• Bachelor of Arts, University of Cambridge - UK, 1994

Research

Research keywords

• Cell division
• Chromosome segregation
• DNA translocation
• Microbiology
• Novel antibiotics
• Site-specific recombination

Research expertise

DNA replication in bacteria: restart of stalled replication forks

The chromosome of E. coli is a circular DNA molecule which is replicated bi-directionally from a single origin (OriC). Multiple copies of the tetracycline operator (tetO) have been placed in the chromosome 16kb to one side of the origin of replication. Expression of a fluorescent tetracycline repressor (TetR-YFP) allows direct visualization of this region in a fluorescence microscope. Using this system, the origin can effectively be followed during duplication and on through the cell cycle. It was found that overexpression of tetR led to cell inviability. The viability of the cells could be recovered by addition of the effector molecule, anhydrous tetracycline (AT) which reduces the binding of TetR to tetO.

Analysis of replication in cells overexpressing TetR showed that the array formed an effective block to replication forks. 2-D gel analysis shows that replication forks can proceed fewer than 500 bp into the tetO array before stalling occurs. Further it is seen that addition of AT leads to a very rapid restart of these stalled forks. Restart of the forks was examined in recA and recB mutant strains and it was found that restart occurred with very similar kinetics to those seen in a wild-type background. This has led us to propose that a stalled replication fork is stable in E. coli for a period of at least 2 hours and that restart does not require recombination. Current work is focusing on what happens when the replisome, stalled at the tetO array block, is disassembled, with a view to understanding replication fork restart pathways and kinetics in vivo. Using this system replication restart can be followed in various mutant backgrounds by examination of fluorescent repressor operators, and by using 2-D gels, to effectively dissect in vivo restart pathways.

FtsK: a fast molecular motor

The multifunctional FtsK protein is involved in cell division and DNA segregation in E. coli. The C-terminal portion of this large protein forms a hexameric ring with the ability to translocate DNA at speeds of ~ 5kb/sec. The FtsK protein is loaded on DNA in a specific orientation by interactions with polarized sequences on the chromosome which ensure that the protein will subsequently move towards the dif site located in the terminus of the chromosome. Once there, FtsK also interacts with the site-specific recombinase XerD to promote recombination between two dif sites. Further, as a result of translocation the two recombining dif sites are brought together in a topologically simple manner so that recombination leads to a simplification of topology, and eventually chromosome unlinking. Using a variety of biochemical techniques the mechanism of directed loading upon DNA, DNA translocation and activation of recombination within a specific synapse topology is being investigated. Using covalently linked multimers of the translocase protion of the protein, hexameric rings can be formed within which mutations can be targeted to specific subunits. This allows more defined analysis of the mechanism of loading and translocation than would otherwise be possible.

Languages

• English

Fields of Research

Centres and Groups

Centre

• PRC - Priority Research Centre for Chemical Biology

Group

Memberships

Body relevant to professional practice.

• Member - Australian Society for Microbiology

Appointments

Future Fellow ARC (Australia)

01/12/2012

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Administrative

Administrative expertise

Member of Institutional Biosafety Committee

Member of Faculty Research and Research Training Committee

Convenor of Biological Sciences seminar series.

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Teaching

Teaching keywords

• BIOL2010
• BIOL2230
• BIOL3100
• CHEM3550

Teaching expertise

Present course taught:

BIOL2010- Biochemistry

CHEM3550 Medicinal Chemistry

BIOL3100 Microbiology

BIOL2230 Biomolecules

Courses lectured at the University of Oxford, Department of Biochemistry:

Modern Molecular Biology: Methods

DNA: Replication and Recombination