Rajavel- Problem Set 2 Q4 completed and graded (Page 2 of 2) 4b) 5’ GTAGCTCGCTGAT 3’ 3’ CATCGAG 5’ No. DNA polymerase cannot extend the bottom strand since there is no primer:template junction and the 3’ end is not available; in other words, there is no ssDNA attached to a shorter primer dsDNA or a free 3’ hydroxyl group end for elongation to occur by DNA polymerase. If DNA were synthesized in the 3’ to 5’ direction, energy would have to come from the growing 5’ strand rather than from free nucleotides (and high energy triphosphates). Once a nucleotide is added, a triphosphate is lost and a single phosphate will stay on the backbone between the new nucleotide and the rest of the strand. In the event of a mismatched nucleotide, the growing strand would be stuck and stop growing. Removal of that nucleotide would result in DNA strand terminated by a monophosphate at the end of the growing strand rather than a much-needed triphosphate (Lehninger, 1973). c) 5’ GTAGCTCGCTGAT 3’ 3’ CGACTA 5’ Yes, a DNA extension will occur since the template strand has a free 3’ hydroxyl group to continue DNA synthesis via elongation. DNA polymerase will extend the 3’ end by adding complementary nucleotides that match to the template strand one at a time forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments are sealed by DNA ligase. d) Circular single stranded DNA DNA Pol I uses double stranded DNA as a substrate in the replication and repair processes. In this case, no primer and no template and hence no synthesis. e) Circular double stranded DNA Yes, an initiator protein nicks one strand of the double-stranded- called the double stranded origin site (DSO). As the initiator protein remains bound to the 5’ phosphate end, the 3’ hydroxyl end is free to serve as primer for DNA synthesis by DNA polymerase III. The nicked strand is then displaced as a single stranded DNA copy; these linear copies can eventually be converted to ds circular DNA. An example of this is DNA replication of SV40/polyoma occurring in the nucleus where T-antigen is required for DNA replication and binds to origin of replication. Polyoma viruses use the host DNA polymerase- it recognizes the viral origin of replication if the T-antigen is present. DNA replication is bidirectional with two replication forks per circular genome with leading and lagging strands, Okazaki fragments, DNA ligase, etc (Hunt, 2010). Dr. R. Notes: First step, a new nick must be introduced for the Polymerase to bind andinitiate synthesis. The absence of a gap to be filled, DNA Pol I will not bind to this molecule and hence no synthesis. The situation is similar to 4a. References: Feng, Hong. (2007). Mutational analysis of bacterial NAD+ dependent DNA ligase: role of motif IV in ligation catalysis. Avcta Biochemica et Biophysisca Sinica. 39(8): 608-616. Hunt, Margaret. (2010). University of South Caroline School of Medicine: Microbiology and Immunology On-line. Virology. Virology: Chapter three DNA virus replication strategies. Retrieved 24 FEB 2014 from http://pathmicro.med.sc.edu/mhunt/dna1.htm Lehninger, A. (1973). Bioenergetics. W.A.Benjamin, Inc. Parker, Jane. (2009). Annual plant reviews, volume 34: molecular aspects of plant disease resistance. Markano Print Media Pte Ltd: Singapore. p.