CH391L/S2013 Taylor Pursell Feb 27 2013
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Identification of Early Replicating Fragile Sites that Contribute to Genome Instability. Barlow JH, Faryabi RB, Callén E, Wong N, Malhowski A, Chen HT, Gutierrez-Cruz G, Sun HW, McKinnon P, Wright G, Casellas R, Robbiani DF, Staudt L, Fernandez-Capetillo O, Nussenzweig A. Cell. 2013 Jan 22. pii: S0092-8674(13)00008-1.
Replication and Fragile Sites
The eukaryotic genome is divide into regions which are programmed to replicate at different times during S-phase of the cell cycle. Gene products important to the cell, or active genes, are replicated in the first half of s-phase, or are associated with early replication, while inactive genes are replicated during the second half of S-phase and are associated with late replication site firing .
Chromosomal fragile sites which are specific stretched of genetic material that are prone to gaps and breaks following replication leading to chromosome instability; these fragile sites are put into two classes: rare and common fragile sites. Rare fragile sites are those found in only a few individuals and are usually associated with genetic disorders (e.g. Fragile X syndrome) while common fragile sites (CFS) are found in all individuals . Common fragile sites are can be associated with large genes, large AT rich regions, and stalled DNA replication [1,3].
B lymphocytes, or B-cells, originate in the bone marrow. The B-cells are then circulated in the blood and become activated through interactions with an antigen and/or T-cells. In this case we will focus on T-cell dependent activation where B-cells undergo terminal maturation after becoming activated through interaction with an antigen and a T-cell. Once activated, large B-cells undergo rapid proliferation and genomic rearrangements important for expansion and specification of the antibody repertoire [1,4,5]. These genome changes come in two classes:
- Somatic Hypermutationi(SHM) in which point mutations, or more rarely deletion or insertions, are induced in the variable region of immunoglobulin (Ig) genes and their surrounding regions.
- Class Switch Recombination (CSR) where one constant region of Ig is deleted and replaced with another[1,5].
Activation-Induced Cytidine Deaminase and DNA Damage
An enzyme called activation-induced cytidine deaminase (AID) is required for SHM and CSR to occur [5,6]. By deaminating cytosine residues on ssDNA that are exposed during Ig transcription, the enzyme turns the cytosine residue into a uridine. The mismatched uridine is glycosylated and removed by uracil DNA glycosylase (UNG) allowing the apyrimidic endonuclease (APE) to generate the DNA breaks needed for SHM and CSR to occur .
AID has been shown to act in the presence of a particular motif: DGYW (D= any bas but C ; G= guainine; Y= pyrimidine ; W= adenosine or thymidine) . Although this is its primary function, it is not uncommon for it to act on other DNA segments containing motif including oncogenes like c-Myc .
In studies preformed in yeast, origins at which replication fork collaspe occurs in response to stalled replication have been identified . These origins do not line up with "replication slow zones" or the yeast equivalent of CFS found at late replication origins [8,9] suggesting there is another class of fragile sites in yeast.
The researchers in this study identify similar early replicating origins at which DNA lesions are found in B-cells; they termed these sites early replicating fragile sites (ERFSs) .
ChIP and ChIP-Seq
Murian splenic B-cell samples were stimulated with LSP/IL4 to cause the cells to synchronously enter cell cycle. After 22 hours, cells should be in S phase (monitored by FACs). Cells were then treated with 10 mM hydroxyurea (HU) which arrests cells at G1/S phase and then cells were harvested. Cells were crosslinked in order to ensure any protein bound to DNA would remain bound. Cells were sonicated in order to break up cells and to fragment DNA. Somicated chromatin samples are then incubated with an antibody specific to the protein of interest (e.g. RPA, BRCA1, etc.) and magnetic breads specific to the constant region of the antibody used.
Once genetic material is harvested, it was prepared for micro sequencing using Illumina’s protocol for sample preparation and was analyzed using the Genome Analyzer IIx.
Fluorescent in-situ hybridization (FISH) analysis
Fluorescent probes that bind to complementary sequences on chromosomes are used to visualize abnormalities. Fluorescent microscopy can be used to find where the probe is bound. In this paper, blue is DAPI-stained DNA, green is the insert in the BAC (either ERFSs or CFSs) and red is telomeric DNA.
- Barlow, J. H. et al. Identification of Early Replicating Fragile Sites that Contribute to Genome Instability. Cell 152:1-13 (2013)
- Goren A, Cedar H. Replicating by the clock. Nature Reviews Molecular Cell Biology In Press 4: 25-32 (2003)
- Durkin, S. G. & Glover, T. W. Chromosome Fraglie Sites. Annual Reviews Genetic 41: 169-192 (2007)
- Parham, Peter. The Immune System. Garland Science, 2. (2005)
- Durandy, A. Activation-induced cytidine deaminase: a dual role in class-switch recombination and somatic hypermutation. Eur. J. Immunol 33: 2069-2073 (2003)
- Kenter, A. L. & Bhattacharya, P. AID: a very old motif newly recognized. Nature Immunology 5: 1203-04 (2004)