Gene name - disc proliferation abnormal
Cytological map position - 43C
Function - Licensing factor
Symbol - dpa
Genetic map position -
Classification - Zn finger motif - cdc21 homolog - MCM4 homolog
Cellular location - nuclear
In order to locate disc proliferation abnormal (dpa) in what might be described as a roadmap for the process of DNA replication, one first must look at the other points on the map.
These are fairly well known (see the DNA replication site), and include DNA polymerases, a DNA ligase, a single stranded DNA binding protein, a processivity factor called Proliferating cell nuclear antigen, and a Tropoisomerase. But what proteins are needed to trigger initiation of DNA replication, and how do they intersect the other proteins and processes involved? Two sets of factors are required. One factor, called the origin recognition complex (ORC), recognizes origins of replications, and signals to the DNA replication apparatus where to carry out its function. For more information about the ORC in Drosophila, see the ORC2 site. Another factor 'licenses' DNA replication, that is, it gives the go ahead for initiation of DNA replication. Information from other organisms, particularly yeast and Xenopus, has allowed the identification of a group of genes that code for called minichromosome maintenance (MCM) proteins that serve to license DNA replication. MCM proteins regulate initiation of DNA synthesis in a cell cycle dependent fashion.
Two MCM genes are known in Drosophila, Minichromosome maintenance 2 and disc proliferation abnormal. Mutations in either gene cause a similar phenotype: cells of imaginal discs are unable to replicate DNA and therefore cells of imaginal discs are unable to multiply. Apparently enough MCM proteins are available from the mother to carry the embryo through the initial stages of development. DmMCM2 mutants exhibit central nervous system defects, and mutations in both genes cause pupal lethality (Feger, 1995 and Treisman, 1995). The following is a summary of the history and biochemisty of DNA proliferation licensing factor.
The first signal for initiation of DNA replicaton involves replication licensing factor (RLF); this licenses replication origins by putting them into an initiation-competent state. The second signal, S-phase promoting factor (SPF), induces licensed origins to initiate, and in doing so removes the license. RLF was first characterized in Xenopus as a component of cell-free extracts supporting chromosomal DNA replication in vitro. In this system, RLF (the license) and SPF (the initiation signal) are prevented from acting at the same time in two different ways. First, RLF cannot cross the nuclear envelope; it can only license DNA when the nuclear envelop is broken down in mitosis. In contrast, SPF can only initiate DNA replication on licensed DNA within an intact nucleus. Second, the spatial separation provided by the nuclear envelope is reinforced by a temporal separation, as both activities are periodic in the cell cycle, that is, they are subject to regulation by cyclins. RLF is activated abruptly during the metaphase-anaphase transition and decays during interphase, while SPF activity can only be detected during interphase.
RLF of Xenopus can be separated into two essential components, RLF-M and RLF-B, both of which are required for licensing. RLF-M, a fraction containing members of the minichromosome maintenance family, associates with chromatin prior to replication but is removed during replication. RLF-M's reassociation with chromatin requires passage through mitosis. RLF-M requires RLF-B ( an as yet uncharacterized fraction) for binding RLF-M to DNA. Apparently RLF binds to origins of replication, but the basis for this binding has not yet been characterized (Chong, 1996 and references).
All six MCM family members (MCM2, 3, 4, 5, 6 and 7) are homologous proteins forming six MCM evolutionary groups. There is only a single gene from any one group in each species. RLF-M complex in Xenopus consists of three or more MCM proteins including MCM2, 3 and 5. In human cells MCM4 is found associated with MCM2, 3 and 7. It is possible that complexes containing different combinations of MCM proteins might be required for initiation of different classes of replication origin. Unlike in S. cerevisiae, where there appears to be cell-cycle-regulated entry of MCM proteins into the nucleus, MCM proteins in mammalian cells and Drosophila are constitutively nuclear. Despite this, mammalian MCMs show intranuclear cell-cycle variation: they are displaced from chromatin during S phase, and do not re-bind until progression into mitosis. The inability of displaced MCMs to re-bind chromatin is most easily explained by a lack of RLF-B activity (the second component of the licensing system) in the nucleus (Chong, 1996 and references).
A candidate for RLF-B type activity is the Cdc6 protein of S. cerevisiae. Cdc6 protein is synthesized during mitosis. in vivo footprinting techniques have been used to examine protein occupancy of the DNA at the origin core site throughout the budding yeast cell cycle. The protective pattern in G2 cells closely resembles that of the purified Origin recognition complex with DNA; late in mitosis (in anaphase) this protection increases by about 50 base pairs. In striking analogy to the behavior of the MCM complex, this extended footprinting is lost during S phase. This preinitiation footprinting and its half-life are dependent upon the action of Cdc6 protein. Thus Cdc6 protein could be tethering the MCM complex to the origin recognition complex (Botchan, 1996 and references).
What are the functions of the MCM proteins? Each protein has a molecular weight of 80-120kDA and a highly conserved central region. This central region contains a predicted consensus sequence for DNA-dependent ATPases and shows some homology to DNA helicases. As DNA unwinding is expected to be required at replication origins, it is attractive to think that this is the function of MCM proteins (Chong, 1996).
The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the DEAD and DEAH box families ATP-dependent helicases signatures.
The DPA protein is 45% identical to fisson yeast cdc21 [an MCM4 homolog](45% identity), the budding yeast CDC54 [another MCM4 homolog] (44%) , CDC46 [an MCM5 homolog](34%), MCM3 (31%) and mammalian P1 [an MCM3 homolog] (31%) (Feger, 1995 and Chong, 1996). Based on sequence similarity, DPA can be assigned to the CDC21/CDC54/MCM4 branch of the MCM gene family. The highest sequence similarity exhibited between members of this family of genes is found in the middle domain. The N-terminal domain of DPA contains a putative Zn finger. The Zinc finger motif, conserved in cdc21, CDC54 and MCM2, has been shown to be essential for MCM2 function. DPA contains five potential phosphorylation sites for cdc2 protein kinase at its N-terminus and a sixth cdc2 phosporylation site following the Zinc finger motif. CDC21, CDC54 and MCM2 also contain potential cdc2 phosphorylation sites, although the number and location of these sites differ in each case (Feger, 1995).
date revised: 7 March 98
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