Biosynthesis of Nucleic Acids: Replication Feb. 25, 2016 CHEM 281

Replication of DNA  Naturally occurring DNA exists in single- stranded and double-stranded forms, both of which can exist in linear and circular forms  Difficult to generalize about all cases of DNA replication  We will study the replication of circular double- stranded DNA and then of linear double-stranded DNA  most of the details we discuss were first investigated in prokaryotes, particularly E. coli

Flow of Genetic Information in the Cell  Mechanisms by which information is transferred in the cell is based on “Central Dogma”

Prokaryotic Replication  Challenges in duplication of circular double-stranded DNA  achievement of continuous unwinding and separation of the two DNA strands  protection of unwound portions from attack by nucleases that attack single-stranded DNA  synthesis of the DNA template from one 5’ -> 3’ strand and one 3’ -> 5’ strand  efficient protection from errors in replication

Prokaryotic Replication (Cont’d)  Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates  Semiconservative replication: each daughter strand contains one template strand and one newly synthesized strand  Incorporation of isotopic label as sole nitrogen source ( 15 NH 4 Cl)  Observed that 15 N-DNA has a higher density than 14 N-DNA, and the two can be separated by density-gradient ultracentrifugation

Evidence for Semiconservative Replication

Which Direction does Replication go?  DNA double helix unwinds at a specific point called an origin of replication  Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional in most organisms  At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed  There is one origin of replication and two replication forks in the circular DNA of prokaryotes  In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin

Bidirectional Replication

DNA Polymerase Reaction  The 3’-OH group at the end of the growing DNA chain acts as a nucleophile.  The phosphorus adjacent to the sugar is attacked, and then added to the growing chain.

DNA Polymerase  DNA is synthesized from its 5’ → 3’ end (from the 3’ → 5’ direction of the template)  the leading strand is synthesized continuously in the 5’ → 3’ direction toward the replication fork  the lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5’ → 3’ direction, but away from the replication fork  lagging strand fragments are joined by the enzyme DNA ligase

Properties of DNA Polymerases DNA polymerase  There are at least five types of DNA polymerase (Pol) in E coli, three of which have been studied extensively

Function of DNA Polymerase  DNA polymerase function has the following requirements:  all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP  Mg 2+  an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication  DNA-Pol I: repair and patching of DNA  DNA-Pol III: responsible for the polymerization of the newly formed DNA strand  DNA-Pol II, IV, and V: proofreading and repair enzymes

Supercoiling and Replication  DNA gyrase (class II topoisomerase) catalyzes reaction involving relaxed circular DNA:  creates a nick in relaxed circular DNA  a slight unwinding at the point of the nick introduces supercoiling  the nick is resealed  The energy required for this process is supplied by the hydrolysis of ATP to ADP and P i

Replication with Supercoiled DNA  Replication of supercoiled circular DNA  DNA gyrase has different role here. It introduces a nick in supercoiled DNA  a swivel point is created at the site of the nick  the gyrase opens and reseals the swivel point in advance of the replication fork  the newly synthesized DNA automatically assumes the supercoiled form because it does not have the nick at the swivel point  helicase, a helix-destabilizing protein, promotes unwinding by binding at the replication fork  single-stranded binding (SSB) protein stabilizes single- stranded regions by binding tightly to them

Primase Reaction  The primase reaction  RNA serves as a primer in DNA replication  primer activity first observed in-vivo.  Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence  Synthesis and linking of new DNA strands  begun by DNA polymerase III  the newly formed DNA is linked to the 3’-OH of the RNA primer  as the replication fork moves away, the RNA primer is removed by DNA polymerase I

Replication Fork General Features

Summary of DNA Replication in Prokaryotes

Proofreading and Repair  DNA replication takes place only once each generation in each cell  Errors in replication (mutations) occur spontaneously only once in every 10 9 to base pairs  Can be lethal to organisms  Proofreading - the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication  Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 10 4 to 10 5 base pairs

Proofreading Improves Replication Fidelity  Cut-and-patch catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides  Nick translation: Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity  Mismatch repair: enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again  Base excision repair: a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

DNA Polymerase Repair

Mismatch Repair in Prokaryotes

Why T and not U?

DNA Recombination  Genetic Recombination- When genetic information is rearranged to form new associations  Homologous - Reactions between homologous sequences  Nonhomologous- Different nucleotide sequences recombine

Recombination

Eukaryotic Replication  Not as understood as prokaryotic. Due in no small part to higher level of complexity.  Cell growth and division divided into phases: M, G 1, S, and G 2

Eukaryotic Replication  Best understood model for control of eukaryotic replication is from yeast.  DNA replication initiated by chromosomes that have reached the G 1 phase

Eukaryotic DNA Polymerase  At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively)

Structure of the PCNA Homotrimer  PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (  ).

The Eukaryotic Replication Fork  The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in Table 10.5.

The Eukaryotic Replication Fork

Telomerase and Cancer  Replication of linear DNA molecules poses particular problems at the ends of the molecules  Ends of eukaryotic chromosomes called telomeres  Telomere- series of repeated DNA sequences

Telomerase and Cancer