Lecture 2: Dna Replication

DNA REPLICATION

DNA REPLICATION Ds nature of DNA provide a means of replication during cell division. Since the separation of the two DNA strands allows complementary strands to be synthesised upon them. Many enzymes and accessory proteins are required for in vivo replication. How about in vitro? In prokaryotes it begins at a region of the DNA termed the origin of replication.

DNA REPLICATION DNA has to be unwound before any of the proteins and enzymes needed for replication can act, and this involves separating the double-helical DNA into single stands. This separating process is carried out by the enzyme DNA helicase. To prevent single strands from re-annealing, small proteins termed single-stranded DNA bindingproteins (SSBs) attach to the single DNA strands

DNA HELICASE PRIES APART THE DOUBLE HELIX(AT A RATE OF 1000 BP/SEC)

(Note: eukaryotic helicase has 6 different subunits)

DNA REPLICATION

SSB HOLDS DNA STRAND BY SUGAR-PHOSPHATE BACKBONE

SINGLE-STRAND DNA BINDING PROTEINS

INITIAL EVENTS AT THE REPLICATION FORK INVOLVING DNA UNWINDING

DNA REPLICATION IS SEMICONSERVATIVE (FOLLOW THE YELLOW STRANDS)

DNA Topoisomerase I: Cut & Swivel (to relaxe wound DNA)

DNA REPLICATION On each exposed single strand a short, complementary RNA chain termed a primer is first produced, using the DNA as a template. The primer is synthesised by an RNA ploymerase enzyme known as a primase, which uses ribonucleoside triphosphate. Then DNA polymerase III (DNA pol III) also uses the original DNA as a template for synthesis of a DNA strand, using the RNA primer as a starting point.

DNA REPLICATION The primer is vital since it leaves an exposed 3’ hydroxyl group for the incoming new nucleotides to be added by DNA polymerase III only to the 3’ end and not the 5’ end of a nucleic acid. Synthesis of the DNA strand therefore occurs only in a 5’ to 3’ direction from the RNA primer. This DNA strand is usually termed the leading strand and provides the means for continuous DNA synthesis.

DNA REPLICATION (c)Ds DNA separates at the origin of replication. RNA polymerase synthesises short RNA primer strands complementary to both DNA strands.

DNA REPLICATION (b) DNA polymerase III, synthesises new DNA strands in a 5’ to 3’ direction, complementary to the exposed, old DNA strands, and continuing from the 3’ end of each RNA primer.

DNA REPLICATION (c) As the replication fork moves away from the origin of replication, DNA polymerase III continues the synthesis of the leading strand, and synthesises DNA between RNA primers of the lagging strand.

DNA REPLICATION (d) DNA polymerase I removes RNA primers from the lagging strand and fills the resulting gaps with DNA. DNA ligase then joins the resulting fragments, producing a continuous DNA strand.

DNA REPLICATION Consequently, DNA synthesis is in the same direction as DNA replication for one strand (the leading strand) and in the opposite direction for the other (the lagging strand). RNA primer synthesis occurs repeatedly to the allow the synthesis of fragments of the lagging strand.

DNA REPLICATION Since the two strands of double helical DNA are antiparallel, only one can be synthesised in a continuous fashion. Synthesis of the other strand must take place in a more complex way. The precise mechanism was worked out by Reiji Okazaki in the 1960s. Here, the strand, usually termed the lagging strand, is produced in relatively short stretches of 1-2 kb termed Okazaki fragments in a 5’ to 3’ direction, using many RNA primers for each individual stretch.

SLIDING CLAMP HOLDS POLYMERASE ONTO DNA (NOTE THAT THIS OCCURS REPEATEDLY ON LAGGING STRAND)

DIMERIC STRUCTURE

LEADING STRAND, LAGGING STRAND, OKAZAKI FRAGMENTS