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UvrD, also known as Helicase II, is one of many components responsible in repairing DNA damage. Helicases use energy from ATP to unwind double helices in metabolic pathways using nucleic acids. ATP molecules are typically used to store energy shared between phosphate groups that gets released when breaking bonds to drive catabolic reactions. Helicases were found in the 1970’s to be DNA-dependent ATPases, meaning that they use ATP hydrolysis to complete its interactions with the different types of nucleic acids it comes into contact with. Helicase II, also called UvrD is the founding member of SF1, one group of six superfamiliies used to identify helicases. SF1 and SF2 members share seven conserved sequence motifs that are involved in ATP Binding [1]. UvrD is important in replication, recombination, and repair from ultraviolet damage and mismatched base pairs. Nucleotide excision repair in a normal cell is supposed to correct pyrimidine dimers and other DNA lesions when bases are displaced from their normal positions. UvrD pairs up with the UvrABC endonuclease system, which works to displace the DNA. This is then repaired by PolI and DNA ligase [2]. UvrD MotifsThere are for UvrD, which are conserved in other homologous structures. The homologous structures mentioned are Helicase 2 homologs, which appear in different species. These conserved motifs are important to maintain the function of UvrD. There are 4 domains that these motifs fit into (not shown). The domains are 1A, 1B, 2A, and 2B. Motifs I, Ia, II-VI are involved in ATP binding. Motifs Ia, III, and V are involved in ssDNA binding. Motif IV is reported to be unique in SF1. They found in their paper, seven new sequence motifs conserved among UvrD homologs. They are Ib, Ic, Id, IVb, IVc, Va, and VIa. These conserved residues are involved in DNA binding or domain 1B and 2B interactions [1]. Separation PinThe "" is a part of the 2B domain and is responsible for unwinding the DNA. This uses a 2 step power stroke, one stroke when ATP is bound and another stroke when ADP and Pi are released. The GIG motif and separation pin work together to unwind the DNA and move it out of the way so UvrD can unwind more DNA. The separation pin also prevents ssDNA once unwound from moving backwards and from reannealing. The proposed method is called the wrench-and-inchworm method, which is when the enzyme binds DNA and attaches at different points and then moves 1 nucleotide per ATP molecule.After an ATP molecule is released, UvrD is then ready to proceed forward to the next nucleotide [1]. UvrD Binding Site for ATP analog (AMPPNP)When determining the structure of UvrD, an ATP analog was used. They used an so that the last phosphate can't be cleaved. Using the unhydrolyzable analog is beneficial in locking in the structure to observe.The green ion shown in the ATP analog scene is a Mg2+ ion, which is essential for ATP hydrolysis and interacts with the β and γ phosphates. The magnesium ion is surrounded by essential residues that when altered, have been shown to have reduced ATPase activity [1]. UvrD Binding Site for ATP analog (ADP•MgF3)To capture the UvrD-DNA-ADP complex, a new crystal structure used ADP•MgF3 after NaF was added to help improve crystal growth. This structure is believed to be a more authentic transition state analog, which differs from the AMPPNP analog slightly. The has a , which is a glycerol molecule, which has hydrogen bonding similar to interactions that E566 has to a 3' OH of the ribose. The DNA isn't actually bound in the crystal structure, but can be used to visualize what hydrogen bonding might look like when connected to the backbone in DNA. , which typically would bind to the 3' OH of the ribose of DNA. Another residue, R37 (Not Shown), binds to the 2' OH of ribose, which has weaker hydrogen bonding. This is a structural component that allows UvrD to bind both ATP and dATP[1]. Stop here References
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