Research Interests
We study early events that promote the initiation of DNA synthesis, which occurs at specific sequences termed replication origins. Various genome-wide approaches have identified from 320 to 420 possible replication origins in budding yeast, and there are perhaps 10,000 origins in human cells. The initiation of DNA replication occurs in a temporally distinct manner during G1 and S phase, and no origin initiates replication (fires) more than once per cell cycle to maintain normal diploid content. In G1 phase, each origin assembles approximately 40 polypeptides in a temporally defined order, culminating in the initiation of DNA replication at the G1/S phase boundary. The first stage of this process is called pre-replicative complex assembly and requires the origin recognition complex (ORC), Cdc6, and Cdt1. ORC directly binds to origin sequences and then recruits Cdt1 and Cdc6 during G1 phase. These three proteins cooperate to load the MCM DNA helicase at origins in an ATP-dependent reaction. Cyclin-dependent kinases and the Cdc7-Dbf4 kinase then catalyze the association of additional proteins with the MCM helicase to activate it, ultimately causing unwinding of the duplex DNA and the initiation of bidirectional DNA synthesis (Figure 1). In our lab we study three key aspects of DNA replication:
- Replication origin structure
- How Cdc6-ATP functions to load the MCM helicase within a chromatin context
- How Cdc7-Dbf4 kinase contributes to the normal cell cycle and human malignancies
Studies to understand the basic molecular biology of DNA replication and cell cycle progression are highly relevant for cancer biology, given that malignant cells often contain mutations in the cell growth and checkpoint pathways that drive normal proliferation. Here we describe recent studies on the Cdc7-Dbf4 kinase, which is a crucial regulator of DNA replication in all eukaryotic cells.
Cdc7-Dbf4 is a conserved, two-subunit, serine/threonine protein kinase that catalyzes DNA synthesis at individual replication origins. Cdc7-Dbf4 promotes DNA synthesis after MCM helicase loading at the origin, likely by activating its helicase activity. This leads to origin unwinding and the assembly of DNA polymerases that initiate bidirectional DNA synthesis. Although Cdc7 is a member of the protein kinase superfamily, it requires the Dbf4 regulatory subunit to activate its kinase activity. We have determined the regions of Dbf4 that bind to and activate Cdc7 kinase by mutational analysis, and we are also investigating how Dbf4 targets Cdc7 kinase to its various substrates in the cell.
In Figure 2, we summarize our analysis of Dbf4 functional regions. The N-terminal third of Dbf4 is dispensable for DNA replication. However, the N-terminus encodes several functions that are important for Dbf4 function. There are two putative classic nuclear localization sequences (NLSs) at 55-61 and 251-257. Although the first sequence is dispensable, deletion of both sequences is lethal. Functionality is restored to a dbf4 mutant lacking the first 265 residues by addition of a heterologous NLS from the SV40 large T-antigen, suggesting that there are no NLS sequences in the Dbf4 C-terminus.
We also identified a BRCT-like domain from residues 115-219 that is apparently conserved in all Dbf4 orthologs. BRCT domains are often present in DNA damage-responsive proteins and they interact with phosphorylated residues. Although Dbf4 mutants deleting this region are viable, they exhibit a slow S-phase and defects in response to DNA-damaging agents such as hydroxyurea, bleomycin, and methylmethane sulfonate. Whether the BRCT-like region governs a DNA repair function for Dbf4 is uncertain, because addition of an SV40 NLS to the dbf4-ND221 deletion mutant reverses most of these DNA replication and damage phenotypes. This suggests that the damage sensitivity is a secondary consequence of lowered nuclear localization and, therefore, compromised initiation activity. Consistent with this explanation, we found that many initiation mutants also exhibit secondary DNA damage sensitivities. Interestingly, deletion of the BRCT-like domain causes defects in late-origin activation, but early origins are activated normally. This raises the intriguing possibility that the BRCT-like domain targets the kinase to late replication origins.
We also constructed a series of C-terminal deletion Dbf4 mutants; such mutants that remove a conserved Zn-finger motif are viable. This indicates that C-terminal residues are not essential for Dbf4 activity. However, deletion of C-terminal residues results in a markedly slower S-phase progression, temperature sensitivity, and DNA damage sensitivity. This suggests that the C-terminus is required to activate full Cdc7 kinase activity or to target it to important replication substrates. Using recombinant Cdc7 and Dbf4 proteins, we found that Dbf4 mutants lacking the C-terminus have a profound defect in Cdc7 kinase activation. The Zn-finger motif also interacts with Cdc7 via a two-hybrid assay, and this interaction depends on conserved residues in the Zn-finger. Lastly, there is a second Cdc7 binding site that overlaps motif M. We found that either Cdc7 binding region could be deleted individually and still allow Cdc7 binding, but deletion of both domains does not allow Cdc7 binding.
We would like to identify proteins that interact with the Dbf4 N-terminus and determine the functional consequences of those interactions. Clearly the N-terminal third of Dbf4 is not required for DNA replication, but these residues are conserved in mouse and human cells and so must confer some critical function. Using a two-hybrid approach, we found that the Dbf4 N-terminus interacts with Polo kinase, a key regulator of mitotic progression. Detailed analysis of this interaction suggests that Dbf4 influences chromosome segregation, which represents a totally new activity for Cdc7-Dbf4 kinase.
