Understanding DNA Replication Machinery in Cell Biology

1. Topic 6 1

2. Topic 6 2 DNA lacks the ability to perform the process of duplication alone The machinery of the cell is required DNA strands are complementary Each contains the information to replicate the alternate strand

3. Topic 6 3 Semiconservative Replication Each daughter duplex is composed of one parent strand and one which is newly synthesized

4. Topic 6 4

5. Topic 6 5 The machinery of replication Replication forks Points at which each of the replicated segments come together Each is a site where Parental double helix strands are separating Nucleotide incorporated into new complementary strands

6. Topic 6 6 The machinery of replication Some Enzyme Classes DNA helicase – unwinds DNA strands Single strands are stabilized by single strand DNA binding proteins DNA topoisomerase – relieves mechanical strain induced by unwinding DNA polymerases – synthesize new strands DNA ligase – forms new bonds between adjacent nucleotides

7. Topic 6 7 The machinery of replication Properties of DNA polymerases Cannot initiate the formation of a new DNA strand Add nucleotides to the 3’ hydroxyl terminus A strand is required to provide the 3’OH Termed a primer All DNA polymerases have two requirements A template DNA strand to copy A primer strand to which nucleotides can be added All add nucleotides from the 5’ to 3’ direction

8. Topic 6 8 The machinery of replication Semidiscontinuous replication OH group at the 3’ end of the primer reacts with the 5’ a-phosphate of the incoming nucleoside phosphate Polymerase molecules on both strands move in the 5’ to 3’ direction One strand grows toward the replication fork The other grows away from the fork

9. Topic 6 9 The machinery of replication Semidiscontinuous replication Strand growing towards the fork Continuous additions of nucleotides to 3’ end Strand growing away from fork Synthesized discontinuously As fragments Before synthesis the fork must move away Once initiated the fragment grows 5’ to 3’ Subsequently each fragment is linked to the next The two daughter strands are synthesized by very different processes

10. Topic 6 10 The machinery of replication Semidiscontinuous replication Strand synthesized continuously – leading strand Strand synthesized discontinuously – lagging strand Okazaki fragments Linked by DNA ligase

11. Topic 6 11 The machinery of replication Semidiscontinuous replication Initiation not via DNA polymerase – by an RNA polymerase – a primase Constructs a short primer of RNA not DNA Required for both strands RNA primers subsequently removed Gaps filled with DNA Sealed by DNA ligase

12. Topic 6 12 The machinery of replication

13. Topic 6 13 The machinery of replication Semidiscontinuous replication DNA polymerase occasionally inserts an incorrect nucleotide DNA polymerase has multiple enzymatic sites – an exonuclease site If an incorrect nucleotide is incorporated Strand tends to bulge Form a single-stranded 3’ terminus Enters the exonuclease site The polymerase stalls allowing the slow-acting exonuclease to excise the incorrect nucleotide

14. Topic 6 14 The machinery of replication Additional features of eurkaryotic cells Incorporate nucleotides into DNA at slower rates Genome replicated in small portions Replicons 50-300 base pairs Replicons close together tend to replicate simultaneously Timing of replication determined by Activity of the genes State of compaction

15. Topic 6 15 The machinery of replication Additional features of eurkaryotic cells Yeast used as a model for eukaryotes Isolation of sequences which promote replication – autonomous replicating sequences (ARSs) Core element – 11 base pairs Binding site for protein complex – origin recognition complex (ORC) Many sites where DNA replication may be initiated Most inhibited by Nucleosome positioning Higher order chromatin structure

16. Topic 6 16 The machinery of replication Additional features of eurkaryotic cells One replication per cycle – control The origin of replication – passage through a series of steps Origin of replication bound by ORC Licensing factors bind assemble the prereplication complex Licensing factors – at least six – Mcm2-Mcm7 Mcm proteins move with the replication fork Mcm proteins are then displaced from DNA but remain in nucleus Mcm proteins cannot reassociate with an origin of replication which has already ‘fired’

17. Topic 6 17 The machinery of replication Additional features of eurkaryotic cells Chromatin structure Nucleosomes and the replication fork Histones – H3H4 tetramers remain intact and are distributed between the daughter duplexes Old and new H3H4 tetramers found on each duplex H2A/H2B dimers – separate and bind randomly to H3H4 tetramers already in place

18. Topic 6 18 DNA is susceptible to damage Ionizing radiation – breaks the backbone of the structure Metabolites – alter base structure UV Radiation – adjacent pyrimidines dimerise Mutations affect germ cells modified trait passed on Mutations affect somatic cells malignant transformation aging

19. Topic 6 19 DNA is susceptible to damage Huge variety of repair mechanisms to repair DNA Proteins patrol DNA searching for alterations and distortions Most repair systems – excise the damaged section DNA duplex – each strand contains the information required for constructing its partner

20. Topic 6 20 Nucleotide Excision Repair (NER) A ‘cut and patch’ mechanism Removes bulky lesions Pyrimidine dimers Chemical groups attached Two pathways Transcription coupled pathway Global pathway

21. Topic 6 21 Base Excision Repair (BER) Initiated by a DNA glycosylase Recognizes alteration Removes base by cleavage of glycosidic bond between the base and deoxyribose Several specific types of DNA glycosylase – for specific modifications Uracil formed from hydrolytic removal of amino group of cytosine Formation of 8-hydroxyguanine – by oxygen free radicals Formation of 3-methyl adenine Following removal of the base – remaining deoxyribose phosphate is removed by endonuclease / phosphodiesterase Gap filled via DNA polymerase and sealed by DNA ligase

22. Topic 6 22 Base Excision Repair (BER)

23. Topic 6 23 Mismatch Repair Mismatch causes distortion of helix Recognized by a repair enzyme Repair system recognizes newly synthesized strand New strand recognized by presence of breaks

24. Topic 6 24 Postreplication Repair Blockage of DNA polymerase progression by pyrimidine dimers and other lesions Replication can be restarted by synthesis of an Okazaki fragment