Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae.

CDC7 and DBF4 encode the essential Cdc7-Dbf4 protein kinase required for DNA replication in eukaryotes from yeast to human. Cdc7-Dbf4 is also required for DNA damage-induced mutagenesis, one of several postreplicational DNA damage tolerance mechanisms mediated by the RAD6 epistasis group. Several genes have been determined to function in separate branches within this group, including RAD5, REV3/REV7 (Pol zeta), RAD30 (Pol eta), and POL30 (PCNA). An extensive genetic analysis of the interactions between CDC7 and REV3, RAD30, RAD5, or POL30 in response to DNA damage was done to determine its role in the RAD6 pathway. CDC7, RAD5, POL30, and RAD30 were found to constitute four separate branches of the RAD6 epistasis group in response to UV and MMS exposure. CDC7 is also shown to function separately from REV3 in response to MMS. However, they belong in the same pathway in response to UV. We propose that the Cdc7-Dbf4 kinase associates with components of the translesion synthesis pathway and that this interaction is dependent upon the type of DNA damage. Finally, activation of the DNA damage checkpoint and the resulting cell cycle delay is intact in cdc7Delta mcm5-bob1 cells, suggesting a direct role for CDC7 in DNA repair/damage tolerance.

Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells.

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (approximately 50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.