Lecture 24: DNA replication. Lecture Outline. Chromosomes. Review

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roofreading Mutation Special problems at the ends of chromosomes nm Review hromosomes Watson-rick model of D structure Base composition ratios =;

1 Lecture 4: D replication Lecture Outline Figure 6.7a, c nm (c) Space-filling model Overview of D replication D has polarity (5, 3 O) Rules of Replication Details of the replication machine roofreading Mutation Special problems at the ends of chromosomes nm Review hromosomes Watson-rick model of D structure Base composition ratios =; = Radioactive labeling experiments show that D is the genetic material Replication is semi-conservative nm Bacterial chromosomes tend to be circular, eukaryotic chromosomes linear D plus associated proteins = chromatin Euchromatin vs heterochromatin

D Structure nm O 3 Sugar denine () O Sugar hymine () O Sugar 5 arbon

8 uanine () O Sugar ytosine () 3 arbon has O In D replication he

basepairing rules (a) he parent molecule has two complementary s of D.

(c) Each parental now serves as a template that determines the order

2 D Structure nm O 3 Sugar denine () O Sugar hymine () O Sugar 5 arbon has O 4 Figure 6.8 uanine () O Sugar ytosine () 3 arbon has O In D replication he parent molecule unwinds, and two new daughter s are built based on basepairing rules (a) he parent molecule has two complementary s of D. Each base is paired by hydrogen bonding with its specific partner, with and with. (b) he first step in replication is separation of the two D s. (c) Each parental now serves as a template that determines the order of nucleotides along a new, complementary. (d) he nucleotides are connected to form the sugar-phosphate backbones of the new s. Each daughter D molecule consists of one parental and one new. Figure 6.9 a d

Replication overview Must maintain integrity of the D sequence through successive rounds of replication (think of the game telephone ) eed to: unwind D, add an R primer, find an appropriate base, add

3 Replication overview Must maintain integrity of the D sequence through successive rounds of replication (think of the game telephone ) eed to: unwind D, add an R primer, find an appropriate base, add it to the growing D fragment, proofread, remove the initial primer, fill in the gap with D, ligate fragments together ll of this is fast, about 00 bp/second Elongating a ew D Strand D polymerases, add nucleotides to the end of a growing ew emplate end end Sugar hosphate Base O animation from Di Figure 6.3 ucleoside triphosphate end yrophosphate O emplate O O 3 Rules of Replication. Semi-conservative. rimer is required 3. emplate is required 4. Elongation occurs 5 -> 3 5. Semi-discontinuous 6. Bidirectional rimer with a free 3 O 3

eeds a template Must add a base to an existing 3 O D synthesis his end has a 5 phosphate ircular bacterial chromosomes have one origin Eukaryotic chromosomes have hundreds or even

5 µm Bubble Replication fork his end has a 3 O wo daughter D molecules eter J. Russell, ienetics: opyright earson Education, Inc., publishing as Benjamin ummings.

arental D Okazaki fragments D pol III emplate Leading Lagging 3 One new, the leading, can elongate continuously as the replication fork progresses.

4 eeds a template Must add a base to an existing 3 O D synthesis his end has a 5 phosphate ircular bacterial chromosomes have one origin Eukaryotic chromosomes have hundreds or even thousands of replication origins Origin of replication arental (template) Daughter (new) lose-up of a replication fork 0.5 µm Bubble Replication fork his end has a 3 O wo daughter D molecules eter J. Russell, ienetics: opyright earson Education, Inc., publishing as Benjamin ummings. Synthesis of leading and lagging s during D replication Model for the replication machine, or replisome D pol Ill elongates D s only in the direction. arental D Okazaki fragments D pol III emplate Leading Lagging 3 One new, the leading, can elongate continuously as the replication fork progresses. 3 he other new, the lagging must grow in an overall direction by addition of short segments, Okazaki fragments, that grow (numbered here in the order they were made). 4 D ligase joins Okazaki fragments by forming a bond between their free ends. his results in a continuous. Figure 6.4 emplate D ligase Overall direction of replication 4

Model for the replication machine, or replisome Detail of the lagging rimase joins R nucleotides into a primer. emplate D pol III adds D nucleotides to the primer, forming an Okazaki fragment.

5 Model for the replication machine, or replisome Detail of the lagging rimase joins R nucleotides into a primer. emplate D pol III adds D nucleotides to the primer, forming an Okazaki fragment. R primer 3 fter reaching the next R primer (not shown), D pol III falls off. 4 fter the second fragment is primed. D pol III adds D nucleotides until it reaches the first primer and falls off. Okazaki fragment 5 D pol replaces the R with D, adding to the end of fragment. 6 D ligase forms a bond 7 he lagging between the newest D in this region is now and the adjacent D of complete. fragment. Figure 6.5 Overall direction of replication eter J. Russell, ienetics: opyright earson Education, Inc., publishing as Benjamin ummings. First look at leading : elicase unwinds a bit of D rimase adds an R primer olymerase adds complementary nucleotides to 3 O and slides down the molecule, continuing to extend the sugar-phosphate chain. (E. coli has about 40,000 turns in the circular chromosome-- each of them must be unwound to separate the D s) ow look at lagging an t form continuous molecule, since synthesis only happens 5 to 3. SO, need many primers and lots of short fragments rimase adds an R primer and olymerase adds nucleotides until it reaches the previous primer nother polymerase removes R primer on adjacent stand and adds ds Ligase connects the fragments 5

6 able 6. Bacterial D replication proteins and their functions ry this at home: Draw a replication bubble, label the bases on the template, primer, and newly synthesized D using the shorthand D notation. onvince yourself that synthesis must go 5 to 3 and that replication must be semi-discontinuous. 6

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