(1) Mapping Replication Origins
We have developed methods to identify origins of replication in order to investigate structural features that define them. Are there specific regions and sequences where DNA synthesis will start? Our lab has devised the Replication Initiation Point (RIP) method to map the start site of DNA replication at the nucleotide level (Bielinsky and Gerbi, Science). Application of this method to yeast (Bielinsky and Gerbi, Molecular Cell) and higher organisms (Bielinsky et al., Current Biology) revealed that DNA synthesis starts directly next to the binding site for the Origin Recognition Complex of six polypeptides. We developed the use of λ-exonuclease (λ-exo) to enrich replicating DNA, and this method is now widely used by many groups. λ-exo is the cornerstone for the popular protocol of nascent strand sequencing (NS-seq) to discover and map origins genome-wide, as recently published by the Gerbi lab with suggested refinements (Foulk et al, Genome Research). Genome-wide identification of replication origins may elucidate their defining features.
Selected Papers
Bielinsky A-K and Gerbi SA (1998). Discrete start sites for DNA synthesis in the yeast ARSI origin. Science 279: 95-98. PMID: 9417033.
Bielinsky A-K and Gerbi SA (1999). Chromosomal ARSI has a single leading strand start site. Mol. Cell 3: 477-486. PMID: 10230400.
Bielinsky A-K, Blitzblau H, Beall EL, Ezrokhi M., Smith HS, Botchan MR and Gerbi SA (2001). Origin recognition complex binding to a metazoan replication origin. Curr. Biol. (Cell Press) 11: 1427-1431. PMID: 11566101.
Foulk MS, Urban JM, Casella C and Gerbi SA (2015) Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res 25: 725-735. PMID: 25695952; PMCID: PMC4417120.
(2) Re-Replication Leading to DNA Amplification
How is an origin is activated more than once in a single S phase, thereby overriding the normal cellular controls against this? Our studies focus on the DNA puffs of the giant salivary gland chromosomes of the fly Sciara coprophila. DNA puffs represent sites of developmentally regulated intrachromosomal re-replication, resulting in gene amplification. We have mapped the origin of amplification for Sciara DNA puff II/9A by 2-D gels, by a 3-D gel method we developed, and by PCR analysis. The origin contracts from an 8 Kb zone of initiation at pre-amplification to 1 Kb at amplification stage. The left boundary is the same at both stages. The change in position of the right boundary when the origin contracts to 1Kb correlates with the appearance there of RNA polymerase II.
Having studied Sciara DNA puff II/9A in depth, we are now extending our experiments to the other 9 major and 9 minor DNA puffs by sequencing the genome of Sciara using cutting edge technology (including the Oxford Nanopore MinION, PacBio and BioNano Irys) to identify the other DNA puffs by their increased copy number. The data suggest that the receptor for the steroid hormone ecdysone binds adjacent to ORC and may interact with the replication machinery to promote re-replication. Moreover, injection of the steroid hormone, ecdysone, into young Sciara larvae prematurely induces DNA amplification, providing the first example of hormonal regulation of DNA replication. These data may provide a useful paradigm for understanding the initiation of gene amplification that is a hallmark of many cancers in humans. We have developed methodology for transformation in Sciara allowing mutagenesis to test the importance of candidate cis-regulatory elements such as the ecdysone receptor binding site.
Selected Papers
Liang C, Spitzer JD, Smith HS and Gerbi SA (1993) Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A. Genes Dev.7: 1072-1084. PMID: 8504930.
Lunyak VV, Ezrokhi M, Smith HS and Gerbi SA (2002). Developmental changes in the Sciara II/9A initiation zone for DNA replication. Mol. Cell. Biol. 22: 8426-8437. PMID: 12446763. PMCID: PMC139883.
Foulk MS, Liang C, Wu N, Blitzblau HG, Smith H, Alam D, Batra M, and Gerbi SA (2006) Ecdysone induces transcription and amplification in Sciara coprophila DNA puff II/9A. Dev. Biol.299: 151-163. PMID: 16938289.
Yamamoto Y, Bliss J and Gerbi SA (2015). Whole organism genome editing: Targeted large DNA insertion via ObLiGaRe nonhomologous end-joining in vivo capture. G3: Genes/Genomes/Genetics 5: 1843-1847. PMID: 26139843; PMCID: PMC4555220.