Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death

1
Rad51-deficient vertebrate cells accumulate
chromosomal breaks prior to cell death

• BE.109
• Module 1 Day 6
• Manuscript discussion

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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 Dec8(12)571-6. link
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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
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What is the normal function of the RAD51 gene?
Tutt A and Ashworth A (2002) Trends Mol Med
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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.
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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

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What is the purpose of this study?

• To study functional role of Rad51 in the
  maintenance of chromosomal DNA during normal cell
  cycling.

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How can we study the function of Rad51?
- By generating conditional RAD51 mutant clones
tetracycline repressible promoter
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What is the experimental strategy?
What are the neo and bsr resistance genes for?
10
What is Southern blot? Why is it necessary for
this study?
More detailed explanation http//www.accessexcell
ence.org/RC/VL/GG/southBlotg.html
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What is Western blot? Why is it necessary for
this study?
Useful link for blotting analysis
http//lifesciences.asu.edu/resources/mamajis/wes
tern/western.html
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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.
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Cell cycle
The cell cycle is an ordered set of events,
culminating in cell growth and division into two
daughter cells. Non-dividing 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/not
es/
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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.
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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 BrdU-positive 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.
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Bromodeoxyuridine (BrdU) Incorporation
Control (left) versus drug treated (right) effect
on cell cycle.
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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_Collapse_a.swf

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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/e
hc/ehc46.htm