DNA Replication Note

DnA ReplicAtion Replication Process Replication Enzymes Helicase: • Helicase separates the DNA strands to form a replication fork (breaks the hydrogen bonds between complementary base pairs) • Single stranded binding proteins prevent strands re-annealing DNA Gyrase: • DNA gyrase reduces the torsional strain created by helicase • It prevents the DNA from supercoiling as it is being unwound DNA Primase: • DNA primase generates a short RNA primer on each strand • Primers provide an initiation point for DNA polymerase III (DNA pol III can only add nucleotides to 3’-end of a primer) 5’ DNA Gyrase relieves torsion Helicase separates DNA SSB Protein DNA Primase makes primer DNA Polymerase III: • Free nucleotides (dNTPs) line up opposite complementary bases • DNA polymerase III covalently joins free nucleotides together DNA Pol III extends chain (5’ → 3’) Okazaki Fragments: • DNA strands are antiparallel, so replication occurs bidirectionally (replication always occurs in a 5’ → 3’ direction on each strand) • Synthesis is continuous on the leading strand (towards fork) and is discontinuous on the lagging strand (away from fork) • Discontinuous segments are called Okazaki fragments DNA Pol I removes primer DNA Polymerase I: • DNA pol I removes RNA primers and replaces them with DNA DNA Ligase: • DNA ligase covalently joins the Okazaki fragments together 3’ DNA Ligase joins fragments 3’ Leading 5’ Lagging DNA Sequencing Sequencing is a technique by which the nucleotide base order of a DNA sequence is elucidated (typically via Sanger method) • Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form covalent bonds (they terminate replication) • Four PCR mixtures are prepared – each with stocks of normal bases and one dideoxynucleotide (ddA, ddT, ddG, ddC) • Whenever the dideoxynucleotide is randomly incorporated, the DNA sequence is terminated at that base position • Because a complete PCR cycle generates millions of sequences, every base position is likely to have been terminated • These sequences are separated by gel electrophoresis to determine base sequence (according to ascending sequence length) • Automated machines can determine the sequence quickly if fluorescent labeling of the dideoxynucleotides has occurred T A G C T ddT 5’ ddA ddG T C ddC C T G A C T T C G A 3’ PCR T C G T C G A Gel Data G A C T G A AG C T