Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci
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Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci. / Garribba, Lorenza; Wu, Wei; Özer, Özgün; Bhowmick, Rahul; Hickson, Ian D; Liu, Ying.
Methods in Enzymology: Mechanisms of DNA Recombination and Genome Rearrangements: Intersection between Homologous Recombination, DNA Replication and DNA Repair. ed. / Marie Spies; Anna Malkova. Vol. 601 Elsevier, 2018. p. 45-58 (Methods in Enzymology; No. 601).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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TY - CHAP
T1 - Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci
AU - Garribba, Lorenza
AU - Wu, Wei
AU - Özer, Özgün
AU - Bhowmick, Rahul
AU - Hickson, Ian D
AU - Liu, Ying
N1 - © 2018 Elsevier Inc. All rights reserved.
PY - 2018
Y1 - 2018
N2 - Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the mechanism by which common fragile sites (CFSs) drive genome instability, we observed that some DNA synthesis was still occurring in early mitosis at these loci. This curious phenomenon of mitotic DNA synthesis (which we now term "MiDAS") appears to be a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination that has been characterized in detail only in lower eukaryotes. During MiDAS, it is proposed that parts of the human genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. To date, the loci that most depend upon this process are those whose replication can be affected by oncogene-induced DNA replication stress (RS), most notably, CFSs. From our studies, it is clear that the successful completion of MiDAS at CFSs can minimize chromosome missegregation and nondisjunction. Nevertheless, it is still not clear which loci that can undergo MiDAS, whether MiDAS is associated with mutations or genome rearrangements, or whether MiDAS really is a form of BIR. In this review, we describe methods for detecting MiDAS both in prometaphase cells and directly on isolated metaphase chromosomes. In addition, we have included methods for combining MiDAS detection either with immunofluorescence (IF) detection of proteins that are recruited to the MiDAS loci, or with fluorescence in situ hybridization using probes that target specific genomic loci.
AB - Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the mechanism by which common fragile sites (CFSs) drive genome instability, we observed that some DNA synthesis was still occurring in early mitosis at these loci. This curious phenomenon of mitotic DNA synthesis (which we now term "MiDAS") appears to be a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination that has been characterized in detail only in lower eukaryotes. During MiDAS, it is proposed that parts of the human genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. To date, the loci that most depend upon this process are those whose replication can be affected by oncogene-induced DNA replication stress (RS), most notably, CFSs. From our studies, it is clear that the successful completion of MiDAS at CFSs can minimize chromosome missegregation and nondisjunction. Nevertheless, it is still not clear which loci that can undergo MiDAS, whether MiDAS is associated with mutations or genome rearrangements, or whether MiDAS really is a form of BIR. In this review, we describe methods for detecting MiDAS both in prometaphase cells and directly on isolated metaphase chromosomes. In addition, we have included methods for combining MiDAS detection either with immunofluorescence (IF) detection of proteins that are recruited to the MiDAS loci, or with fluorescence in situ hybridization using probes that target specific genomic loci.
KW - Common fragile sites
KW - DNA replication stress
KW - EdU
KW - Fluorescence in situ hybridization
KW - Immunofluorescence
KW - Mitosis
U2 - 10.1016/bs.mie.2017.11.025
DO - 10.1016/bs.mie.2017.11.025
M3 - Book chapter
C2 - 29523241
SN - 978-0-12-813979-0
VL - 601
T3 - Methods in Enzymology
SP - 45
EP - 58
BT - Methods in Enzymology
A2 - Spies, Marie
A2 - Malkova, Anna
PB - Elsevier
ER -
ID: 196436919