Site-specific intra-chromosomal DNA amplification is common in many cancers, but the events that initiate this process are unknown. To address this question, we will use one of only two known systems where this occurs as a normal process in development, namely in the salivary gland polytene chromosome DNA puffs of the fly Sciara. We propose to elucidate the mechanism for induction of DNA puff amplification, making use of our recent advances in development of a method for Sciara transformation, assembly of the Sciara genome, and identification of DNA puffs in the genome sequence. First we will map the origins of replication and re-replication in the salivary gland genome, using NS-seq and ORC-ChIP as well as validation of the re-replication origins by DNA combing. Next, we will identify consensus motifs that map near the amplification origins and test them by deletion mutagenesis as well as a functional test by placement at an origin that normally does not re-replicate to ask if it is now driven to re-replicate. With the cis-regulatory elements in hand, we will identify the trans-acting factors (protein and RNA) that bind to these elements by pull-down experiments with in vivo occupancy validation by ChIP. For a functional test, we will inducibly tether the trans-acting factor to an origin that normally does not re-replicate to ask if it is now driven to re-replicate. This will include development of methodology for induction of tethered RNA, if the trans-acting factor is a ncRNA. In the final section of this grant application, we will use the trans-acting factor for pull-down experiments to identify proteins that interact with it. Among many possibilities, such interacting proteins may be components of the replication machinery or modify it to be continually load and activate Mcms or modify the chromatin environment. Our results will suggest a mechanism for DNA puff amplification, and future experiments can test if this may serve as a paradigm for initiation of DNA amplification in cancer.