Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.     

Papillomavirus E1 Proteins: Form,Function, and Features

The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.     

Mechanism for Aar2p function as a U5 snRNP assembly factor

Little is known about how particle-specific proteins are assembled on spliceosomal small nuclear ribonucleoproteins (snRNPs). Brr2p is a U5 snRNP-specific RNA helicase required for spliceosome catalytic activation and disassembly. In yeast, the Aar2 protein is part of a cytoplasmic precursor U5 snRNP that lacks Brr2p and is replaced by Brr2p in the nucleus. Here we show that Aar2p and Brr2p bind to different domains in the C-terminal region of Prp8p; Aar2p interacts with the RNaseH domain, whereas Brr2p interacts with the Jab1/MPN domain. These domains are connected by a long, flexible linker, but the Aar2p–RNaseH complex sequesters the Jab1/MPN domain, thereby preventing binding by Brr2p. Aar2p is phosphorylated in vivo, and a phospho-mimetic S253E mutation in Aar2p leads to disruption of the Aar2p–Prp8p complex in favor of the Brr2p–Prp8p complex. We propose a model in which Aar2p acts as a phosphorylation-controlled U5 snRNP assembly factor that regulates the incorporation of the particle-specific Brr2p. The purpose of this regulation may be to safeguard against nonspecific RNA binding to Prp8p and/or premature activation of Brr2p activity.     

Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein

Sumoylation of the papillomavirus (PV) origin binding helicase E1 protein is critical for its function. Consequently, factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. We investigated the role played by phosphorylation and two known SUMO E3 ligases, RanBP2 and PIAS proteins, on the sumoylation of E1. E1 sumoylation was unaffected by phosphorylation as both wild-type and pseudo-phosphorylation mutants of BPV E1 exhibited similar sumoylation profiles. RanBP2 bound to BPV E1, but not to HPV11 E1, and lacked sumoylation enhancing activity for either E1. In contrast, proteins of the PIAS family (except PIASy) bound to both BPV and HPV11 E1 and stimulated their sumoylation. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation but was dispensable for their PV E1 binding activity. Miz1, the PIAS protein exerting the strongest E1 sumoylation enhancing activity, favored SUMO1 versus SUMO2 as the modifier and was shown to be transcribed in a keratinocyte cell line. This study indicates PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections.     

ClpB chaperone passively threads soluble denatured proteins through its central pore

ClpB disaggregase forms a ring‐shaped hexamer that threads substrate proteins through the central pore using energy from ATP. The ClpB protomer consists of an N‐terminal domain, a middle domain, and two AAA+ modules. These two AAA+ modules bind and hydrolyze ATP and construct the core of the hexameric ring. Here, we investigated the roles of the two AAA+ modules in substrate threading. BAP is an engineered ClpB that can bind ClpP proteolytic chamber; substrates threaded by BAP are degraded by ClpP. We combined BAP with conserved motif mutations in two AAA+ modules and measured the steady‐state rates of threading of soluble denatured proteins by these mutants over a range of substrate concentrations. By fitting the data to the Michaelis‐Menten equation, k_cat and K_m values were determined. We found that the kinetic parameters of the substrate threading correlate with the type of mutation introduced rather than the ATPase activity of the mutant. Moreover, some mutants having no or marginal ATPase activity could thread denatured proteins significantly. These results indicate that ClpB can passively thread soluble denatured proteins.     

Selective modulation of the functions of a conserved DNA motor by a histone fold complex

Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Elucidating the mechanisms that regulate these motor proteins is central to understanding genome maintenance processes. Here, we show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair. Mechanistically, scMHF relieves the inhibition imposed by the structural maintenance of chromosome protein Smc5 on Mph1 activities relevant to replication-associated repair through binding to Mph1 but not DNA. Thus, scMHF is a function-specific enhancer of Mph1 that enables flexible response to different genome repair situations.     

Dissection of functional domains of the packaging protein of bacteriophage T3 by site-directed mutagenesis

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are joined together in head-to-tail fashion through terminally redundant sequences. During packaging of DNA, mature monomers are cut from the concatemer. The cutting is obligatorily coupled to DNA packaging. The packaging of phage DNA is under the control of a pair of noncapsid proteins, called packaging proteins, gp 18 and gp19. gp19 is an ATP-binding protein that plays multiple roles in DNA packaging. gp19 is predicted, from the sequence of its gene, to contain 586 amino acids, and has consensus sequences for an ATP binding site. To dissect structure-function relationships of gp19, mutations were introduced into the ATP binding domain and the mutant proteins were overproduced, purified and characterized. Mutant gp19 with a Gly-to-Asp mutation at amino acid 61 (gp19 G61D) was defective in DNA packaging due to an altered interaction with ATP. Gp19 G424E, with a change in another putative ATP binding domain, was active in DNA packaging but was defective in DNA cutting. A second mutation in the latter domain, gp19 K430T, and a mutation at 553 (to give gp19 H553L), within a putative Mg2+ binding domain, had only minor effects on gp19 activities.     

FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair

In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.     

Structural basis for the enhancement of eIF4A helicase activity by eIF4G

The eukaryotic translation initiation factors 4A (eIF4A) and 4G (eIF4G) are crucial for the assembly of the translationally active ribosome. Together with eIF4E, they form the eIF4F complex, which recruits the 40S subunit to the 5' cap of mRNA. The two-domain RNA helicase eIF4A is a very weak helicase by itself, but the activity is enhanced upon interaction with the scaffolding protein eIF4G. Here we show that, albeit both eIF4A domains play a role in binding the middle domain of eIF4G (eIF4G-m, amino acids 745-1003), the main interaction surface is located on the C-terminal domain. We use NMR spectroscopy to define the binding site and find that the contact surface is adjacent to the RNA-, ATP-, and eIF4A-NTD-interacting regions. Mutations of interface residues abrogated binding, confirmed the interface, and showed that the N-terminal end of eIF4G-m interacts with the C-terminal domain of eIF4A. The data suggest that eIF4G-m forms a soft clamp to stabilize the closed interdomain orientation of eIF4A. This model can explain the cooperativity between all binding partners of eIF4A (eIF4G, RNA, ATP) and stimulation of eIF4A activity in the eIF4F complex.     

The interaction of SV40 large T antigen with unspecific double-stranded DNA: an electron microscopic study.

T antigen, an early protein encoded by simian virus 40 (SV40), is a specific DNA-binding protein with high affinity for elements in the viral origin of replication where it forms a double-hexameric complex as a prerequisite for DNA untwisting and, in the presence of ATP hydrolysis, for DNA unwinding. Like other specific DNA-binding proteins, T antigen also associates with DNA strands of random sequence albeit at reduced affinity. In addition, T antigen is able to unwind unspecific DNA sequences starting from internal binding sites. This property could be a step in the pathway leading to the chromosomal rearrangements that are frequently observed in SV40-transformed cells. This possibility prompted us to investigate the binding of T antigen to unspecific DNA using electron microscopy. We observed that the protein binds randomly to many unspecific DNA sites excluding a preference for particular DNA sequences or structural features. Addition of ATP to the binding buffer induces the formation of oligomeric, possibly hexameric, T antigen complexes that frequently align to form long arrays of DNA-bound protein. Magnesium salts induce the formation of tightly packed T antigen aggregates which bind to DNA to form many DNA branches and loops that emanate from the aggregated protein core. Upon ATP hydrolysis, aggregated T antigen catalyzes the unwinding of DNA duplices.     

RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block

Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB). The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate. The helicase activity of these proteins was dependent on Mg2+ and ATP. The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex.     

HPV E1 up-regulates replication-related biochemistries of AAV Rep78

Human papillomavirus type 16 (HPV) E1 protein provides helper function for the adeno-associated virus type 2 (AAV) life cycle. E1 is the replication protein of HPV, analogous to AAV Rep78, but without the endonuclease/covalent attachment activity of Rep78. Previously we have shown that E1 and Rep78 interact in vitro. Here we investigated E1's effects on Rep78 interaction with AAV's inverted terminal repeat (ITR) DNA in vitro, using purified Rep78 and E1 proteins from bacteria. E1 enhanced Rep78-ITR binding, ATPase activity, Rep78-ITR-covalent linkage and Rep78-ITR-endonuclease activity (central to AAV replication). These enhancements occurred in a dose-dependent manner whenever assayed. However, overall Rep78-plus-E1 helicase activity was lower than Rep78's helicase activity. These data suggest that E1's broad-based helper function for the AAV life cycle (AAV DNA, mRNA, and protein levels are up-regulated by E1) is likely through its ability to enhance Rep78's critical replication-required biochemistries on ITR DNA.