Eileen L. Beall

DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC–DNA binding

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well‐studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis‐requirements for DmORC:DNA interaction. DmORC displays at best six‐fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA‐binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30‐fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator–initiator interactions and that in vivo other proteins must determine ORCs localization.

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-β, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-β (refs 7–9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

DNA binding by the KP repressor protein inhibits P‐element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P‐element transposition is tightly regulated, and P‐element‐encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild‐type and mutant KP proteins were tested in vitro using several different assays for P‐element transposase activity. These studies indicate that the site‐specific DNA‐binding activity of the KP protein is essential for repressing transposase activity. The DNA‐binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N‐terminal 88 amino acids. Within this region, there is a C2HC putative metal‐binding motif that is required for site‐specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA‐binding sites near the P‐element termini.

Transposase makes critical contacts with, and is stimulated by, single-stranded DNA at the P element termini in vitro

P elements transpose by a cut‐and‐paste mechanism. Donor DNA cleavage mediated by transposase generates 17 nucleotide (nt) 3′ single‐strand extensions at the P element termini which, when present on oligonucleotide substrates, stimulate both the strand‐transfer and disintegration reactions in vitro. A significant amount of the strand‐transfer products are the result of double‐ended integration. Chemical DNA modification–interference experiments indicate that during the strand‐transfer reaction, P element transposase contacts regions of the substrate DNA that include the transposase binding site and the duplex portion of the 31 bp inverted repeat, as well as regions of the terminal 17 nt single‐stranded DNA. Together these data suggest that the P element transposase protein contains two DNA‐binding sites and that the active oligomeric form of the transposase protein is at least a dimer.

humpty dumpty Is Required for Developmental DNA Amplification and Cell Proliferation in Drosophila

The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

Significance The P-element transposon is a mobile DNA that invaded the Drosophila genome approximately 100 y ago. P elements were identified by studying a genetic syndrome called “hybrid dysgenesis.” The elements use their encoded transposase for mobility, but rely on host-cell factors for essential parts of their life cycle. Here we demonstrate biochemically that a Drosophila-encoded bZIP heterodimer binds to the P-element terminal 31-bp sequences. We used genetics to show that these proteins play a role in repairing DNA breaks caused by P-element transposase activity during hybrid dysgenesis and other types of DNA damage. These results provide an example of the mechanisms that the host genome uses to combat genome instability caused by foreign DNA invasion. In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein–DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed “Inverted Repeat Binding Protein (IRBP) 18” and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.