Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery

Abstract

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

Figure 1

Scheme of biochemical assay for measuring unwinding activity of ATP/Mg²⁺‐dependent DNA helicase (A) and autoradiogram of the gel (B). (A) Asterisks denote the ³²P‐labeled end of the DNA. The partial duplex DNA helicase substrate was prepared by annealing the radiolabeled DNA oligo to M13 ssDNA (circular) as described previously [65]. (B) Lane 1, reaction without enzyme; lane 2, heat‐denatured substrate; lane 3, reaction in presence of DNA helicase enzyme. S, Substrate; UD, unwound DNA.

Figure 2

Structures of the linear partial duplex substrates commonly used to determine the direction of translocation of the helicase. The 3′ to 5′ directional substrates are on the left and 5′ to 3′ directional substrates are on the right. Asterisks denote the ³²P‐labeled end.

Figure 3

Interaction of monomeric or oligomeric DNA helicases with the DNA forked substrate. (A) Monomeric helicase binds to both ssDNA and dsDNA. (B) In homodimeric helicases, one subunit always binds to the ssDNA track along which it moves. (C) Heterodimeric helicase contains two separate domains: one subunit binds/interacts with dsDNA and anchors the helicase to the DNA lattice and the other subunit interacts with ssDNA and translocates along it. (D) Hexameric or oligomeric helicases contain a ring‐like structure that enables the proteins to encircle the DNA and thus prevent local reannealing. In this case one or more subunits bind to ssDNA at the ss/dsDNA junction.

Figure 4

Recruitment to activation of Mcm complex during initiation of DNA replication at the origin. (A) The origin is ‘marked’ by the origin recognition complex (orc). (B) Assembly of the prereplication complex (pre‐RC) begins during the G1 phase, when the ‘loading factors’ Cdc6 and Cdt1 are recruited to the replication origin to which orc (and Mcm10) bind. (C) Two Mcm complexes (ring‐shaped hexamers) load on to the origin, which is facilitated by Cdc6 and Cdt1. The Cdc7‐Dbf4 kinase (DDK) is also recruited to the origin during the G1 phase and phosphorylates the Mcm complex during the S phase. (D) The loading factors have been displaced from the DNA; the phosphorylated two Mcm complexes (enzymatically active helicase) have moved apart along the template, generating a replication ‘bubble’ by unwinding and displacing the orc. At each ‘fork’ the Cdc45 protein binds. The loading of other DNA replication factors, such as DNA polymerase α, RPA, and primase, etc., start at the time of initial DNA melting, which leads to the initiation of DNA replication.

Figure 5

The RecQ helicase family. Schematic representation of the RecQ family of DNA helicases from human (WRN, BLM, RECQ4, RECQ5, RECQL), Xenopus (FFA‐1, xBLM), Drosophila (DmBLM), Saccharomyces cerevisiae (Sgs1), Saccharomyces pombe (Rqh1) and E. coli (RecQ). Proteins are aligned by their conserved helicase domains. The size of each protein (in amino acids) is indicated in parentheses under the respective member. The key to various domains is indicated in the box at the bottom. Note: The Drosophila melanogaster RECQ5 and RECQE, Caenorhabditis elegans RecQ5, and RecQ homologs from Arabidopsis thaliana are not shown.