*Values in parentheses are for the highest-resolution shell. This approach was used to obtain high resolution structures of DNAP-I for the starting primer-template complex (n) and two translocated products obtained for the n + 1 and n + 2 nucleotide addition steps using the same primer-template duplex (n) described in previous in crystallo studies ( Figure 1a)( Johnson et al., 2003). Following primer-extension, the enzyme-product complex was crystallized and cocrystal structures of Bst DNAP-I were solved to resolutions of 1.5 – 2.0 Å ( Table 1). In these reactions, the starting enzyme-primer-template complex was incubated with solutions of either buffer, dTTP, or dTTP and dATP for 30 min at 37☌. Recognizing that in crystallo and solution catalyzed enzymatic reactions can produce different structural results with potentially different functional interpretations ( Ehrmann et al., 2017), we chose to investigate the translocated intermediates of DNAP-I using a direct crystallization method that involves solving crystal structures of the enzyme-product complex obtained from primer-extension reactions performed in solution rather than inside the environment of a protein crystal. Together, these structures provide new insight into the mechanism of DNA synthesis and highlight the dynamic nature of the finger subdomain in the enzyme active site. Here we report five crystal structures of DNAP-I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. However, the pre-insertion site has not been witnessed in polymerases with homologous active sites ( Eom et al., 1996 Li et al., 1998 Yin and Steitz, 2002), implying that DNAP-I follows a complex enzymatic pathway that contains numerous intermediates, many of which have not yet been observed in protein crystals. The pre-insertion site is a hydrophobic pocket located between the O and O1 helices of the finger subdomain where the n + 1 templating base resides prior to forming the nascent base pair with the incoming dNTP substrate ( Johnson et al., 2003). The prevailing mechanism invokes the use of a distinct pre-insertion site, observed in the translocated product of in crystallo catalyzed primer-extension reactions where dNTP substrates are soaked into pre-formed crystals of DNAP-I bound to a primer-template duplex ( Figure 1-figure supplement 1)( Johnson et al., 2003 Kiefer et al., 1998). Structural insights into the mechanism of DNA synthesis have been obtained from crystal structures of a thermostable bacterial ( Geobacillus stearothermophilus, Bst) DNAP-I large fragment that retains catalytic activity inside the crystal lattice ( Johnson et al., 2003 Kiefer et al., 1998). IntroductionĭNA polymerase I (DNAP-I) has long been viewed as the canonical model for DNA synthesis in cells ( Lehman et al., 1958). Understanding how polymerases modify their form while making DNA copies could lead to better therapies for diseases in which this process has become faulty, like cancer. uncovered may help scientists develop new biotechnologies and medicines. The intermediate structures that Chim, Jackson et al. This modified method captured different steps in the process and detailed how the enzyme adjusts its structure as it moves along the template strand.
The reaction was then frozen and X-ray crystallography was used to take images. used a particular method for making frozen polymerase crytals by allowing the enzyme to add new bases in liquid form. Yet, to fully understand the mechanisms of DNA synthesis all intermediate structures need to be identified. Scientist often use a technique called X-ray crystallography to study intermediate structures of frozen polymerase crystals as the enzyme constructs DNA. If this process becomes faulty, it can lead to various diseases, including cancer. Every time a new base is added, the polymerases must modify their structures several times. Each strand is copied by adding new bases one at a time. During this process, the helix unwinds and enzymes called polymerases produce new strands (using the old ones as a template). When a cell divides, it needs to make a copy of the DNA, so that each new cell will have an exact replica from the old cell. The two strands are held together by bonds between the bases.
Each strand contains repeating units, with every unit consisting of a phosphate group and a sugar molecule bound to one of four bases. DNA molecules consist of two separate strands that spiral around each other to form a structure called the double helix.
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