Transcript Rad51-deficient vertebrate cells accumulate

Rad51-deficient vertebrate
cells accumulate chromosomal
breaks prior to cell death
BE.109
Module 1 Day 6
Manuscript discussion
What is the normal function of the
RAD51 gene?
The RAD51 gene makes a protein also called RAD51, which is essential for the
repair of damaged DNA. The protein made by the BRCA2 gene binds to and
regulates the RAD51 protein to fix breaks in DNA. These breaks can be caused
by natural or medical radiation. They also occur when chromosomes exchange
genetic material (when pieces of chromosomes trade places) in preparation for
cell division. The BRCA2 protein transports the RAD51 protein to sites of DNA
damage in the cell nucleus. RAD51 then binds to the damaged DNA and encases
it in a protein sheath, which is an essential first step in the repair process. In
addition to its association with BRCA2, the RAD51 protein also interacts with the
protein made by the BRCA1 gene. By repairing DNA, these three proteins play a
role in maintaining the stability of the human genome.
Useful reference:
Tutt A and Ashworth A. The relationship between the roles of BRCA genes in
DNA repair and cancer predisposition. Trends Mol Med. 2002 Dec;8(12):571-6.
[link]
What is the normal function of
the RAD51 gene?
A model of the DNA-damage signal transduction cascade
Tutt A and Ashworth A (2002) Trends Mol Med
What is the normal function of
the RAD51 gene?
Tutt A and Ashworth A (2002) Trends Mol Med
Where is the RAD51 gene located?
The RAD51 gene is located on the long (q) arm of chromosome 15 at
position 15.1. More precisely, the RAD51 gene is located from base pair
38,774,660 to base pair 38,811,645 on chromosome 15.
What other names do people use for
the RAD51 gene or gene products?
• DNA repair protein RAD51 homolog 1
• HRAD51
• HsRAD51
• RAD51A
• RAD51_HUMAN
• RECA
• RecA, E. coli, homolog of
• RecA-like protein
• recombination protein A
What is the purpose of this study?
To study functional role of Rad51 in the
maintenance of chromosomal DNA during
normal cell cycling.
How can we study the function
of Rad51?
- By generating conditional RAD51 mutant clones
tetracycline repressible promoter
What is the experimental
strategy?
What are the neo and bsr resistance genes for?
What is Southern blot?
Why is it necessary for this study?
More detailed explanation:
http://www.accessexcellence.org/RC/VL/GG/southBlotg.html
What is Western blot? Why is it
necessary for this study?
Useful link for blotting analysis:
http://lifesciences.asu.edu/resources/mamajis/western/western.html
What is the cell type the authors
used? Why did they use it?
The chicken B lymphocyte line DT40
- The high level of homologous recombination allowed us to perform targeted
disruption of both RAD51 alleles.
Cell cycle
The cell cycle is an ordered set of
events, culminating in cell growth and
division into two daughter cells. Nondividing cells not considered to be in
the cell cycle. The stages, pictured to
the left, are G1-S-G2-M. The G1
stage stands for "GAP 1". The S
stage stands for "Synthesis". This is
the stage when DNA replication
occurs. The G2 stage stands for
"GAP 2". The M stage stands for
"mitosis", and is when nuclear
(chromosomes separate) and
cytoplasmic (cytokinesis) division
occur.
Image: http://www.bmb.psu.edu/courses/biotc489/notes/
How can we analyze the cell
cycle?
A common way of ascertaining a proportion of a
population of cells at any given part of the cell cycle
is to analyse according to the DNA content (by
definition G2 cells have twice as much DNA as G1).
The process requires cell permeabilisation, RNAse
treatment and staining (most commonly with PI
however other stains such as DAPI and 7AAD are
also used) prior to analysis. Following exclusion of
cell doublets and clumps from the analysis, a
healthy, growing cell population will exhibit a
characteristic histogram profile as above. The
analysis of this histogram by the pragmatic approach
will then ascertain the proportional cell numbers in
G1-S-G2/M phases and can also be used to
measure nuclear condensation in apoptotic cells.
Bromodeoxyuridine (BrdU)
Incorporation
BrdU is an analogue of thymidine
and will be taken up into the DNA
of cycling cells. To detect this, we
can unwind the DNA (by using
acid, alkali or enzyme) and then
use an antibody against BrdU. In
this way, we can separate G1, S
and G2 cells. Obviously BrdUpositive cells will equate to S
phase cells only when the
labeling time is short as cells in
late S at the beginning of the
labeling period.
Bromodeoxyuridine (BrdU)
Incorporation
Control (left) versus drug treated (right) effect on cell cycle.
How is replication related with
homologous recombination?
• Animation file made by Justin H. Lo may
help you understand how replication is
related with homologous recombination:
http://web.mit.edu/jhlo/www/urop/Fork_Col
lapse_a.swf
Isochromatid-type gaps and
breaks
Both sister chromatids of a single chromosome are
broken at the same locus
Useful link: http://www.inchem.org/documents/ehc/ehc/ehc46.htm