LECTURE DNA replication and Polymerases-1.pptx

Molecular genetics lecture on replication and polymerase

1. MIRPUR UNIVERSITY OF SCIENCE AND TECHNOLOGY (MUST), MIRPUR DEPARMENT OF BIOTECHNOLOGY

2. INTRODUCTION TO MOLECULAR GENETICS HND-2302 Lecture: DNA Replication and Polymerases Faculty of Natural and applied sciences Dr Zeeshan Shamim (Assistant Professor) Date: April 23, 2020

3. 3 DNA REPLICATION IS SEMICONSERVATIVE That’s all for DNA replication DNA replication is the process of copying cell’s DNA. Three billion base pairs are accurately copied during division of one of your trillions cells. The basic mechanism of DNA replication in organisms is similar. DNA replication if semiconservative, each strand acts as template for the synthesis of new complementary strand. Thus, two daughter molecules are formed from one starting molecule. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

4. 4 In a cell, DNA needs to be copied accurately so to avoid defective DNA in next cell. To perform the task smoothly, cell uses variety of proteins and enzymes to accomplish the process of replication. In prokaryotes, three types of main DNA polymerases are known: DNA polymerse I DNA polymerase II DNA polymerase III DNA polymerases are responsible for synthesizing DNA by adding nucleotides one by one to the growing chain of DNA. DNA POLYMERASES INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

5. 5 KEY FEATURES OF ESCHERICHIA COLI DNA POLYMERASES A template is always required. DNA polymerases add nucleotides to the 3’ of a DNA strand. A short sequence of oligonucleotide called primer is required to initiate the replication process. They have “proof reading” abilities which are to remove wrong nucleotides which are incorporated accidently during the replication process. DNA polymerase I and III are the major polymerases in E. coli. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

6. 6 DNA POLYMERASE I This is not the primary enzyme of replication, instead it performs clean up functions during replication, repair and recombination. Pol I (109 kDa ) possesses four enzymatic activities: A 5'→3' (forward) DNA-dependent DNA polymerase activity , requiring a 3' primer site and a template strand A 3'→5' (reverse) exonuclease activity that mediates proofreading (deletion of this domain leaves a large Klenow fragment which has polymerization activity) A 5'→3' (forward) exonuclease activity mediating nick translation during DNA repair. A 5'→3' (forward) RNA-dependent DNA polymerase activity . Pol I operates on RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA templates, and this activity is probably of only limited biological significance. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

7. 7 INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim DNA POLYMERASE I 3’-5’ EXONUCLEASE ACTIVITY

8. 8 KLENOW FRAGMENT INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

9. 9 DNA POLYMERASE I 5’-3’ EXONUCLEASE ACTIVITY INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

10. 10 DNA POLYMERASE III Thomas Kornberg INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

11. 11 DNA POLYMERASE III The replisome (multiprotein complex) is composed of the following: 2 DNA Pol III enzymes , each comprising α , ε and θ subunits. (It has been proven that there is a third copy of Pol III at the replisome.) the α subunit (encoded by the dnaE gene) has the polymerase activity . the ε subunit ( dnaQ ) has 3'→5' exonuclease activity . the θ subunit ( holE ) stimulates the ε subunit's proofreading . 2 β units ( dnaN ) which act as sliding DNA clamps, they keep the polymerase bound to the DNA. 2 τ units ( dnaX ) which act to dimerize two of the core enzymes (α, ε, and θ subunits). 1 γ unit (also dnaX ) which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit ( holA ), and 1 δ' subunit ( holB ). The δ is involved in copying of the lagging strand. ( holC ) and Ψ ( holD ) which form a 1:1 complex and bind to γ or τ. X can also mediate the switch from RNA primer to DNA INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

12. 12 DNA POLYMERASE III ACTIVITY DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second . DNA Pol III activity begins after strand separation at the origin of replication. Because DNA synthesis cannot start de novo , an RNA primer , complementary to part of the single-stranded DNA, is synthesized by primase (an RNA polymerase) INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

13. 13 ORIGIN OF REPLICATION ( OriC ) HOW DO DNA POLYMERASES THINK WHERE TO BEGIN REPLICATION DNA replication starts from a very specific region on DNA which is called Origin of replication which is recognized by its specific nucleotides. Most bacteria including E. coli have single A/T rich OriC which is 245 bp in case of E. coli . INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

14. 14 ORIGIN OF REPLICATION ( OriC ) The origin of replication in E. coli is termed oriC ori gin of C hromosomal replication Three types of DNA sequences in oriC are functionally significant AT-rich region ( DnaB box) DnaA boxes GATC methylation sites INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

15. 15 OriC OF E. COLI CHROMOSOME DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication initiation factor which promotes the unwinding of DNA at oriC . DnaB helicase is an enzyme in bacteria which opens the replication fork during DNA replication. dnaC is a loading factor that complexes with the C-terminus of helicase dnaB and inhibits it from unwinding the dsDNA at a replication fork. Single-stranded DNA-binding protein (SSB) binds to single-stranded regions of DNA. It prevents the strands from hardening too early during replication, it protects the single-stranded DNA from being broken down by nucleases during repair, and it removes the secondary structure of the strands so that other enzymes are able to access them and act effectively upon the strands INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

16. 16 PRIMERS AND PRIMASE DNA polymerases can not initiate chain extension by binding on single stranded chain (with some exceptions). They always need DNA-RNA duplex to initiate nucleotide addition process on template strand. This problem is solved by an enzyme primase (encoded by dnaG gene). The enzyme DnaG , and any other DNA primase, synthesizes short strands of RNA known as oligonucleotides during DNA replication. These oligonucleotides are known as primers because they act as a starting point for DNA synthesis. DnaG catalyzes the synthesis of oligonucleotides that are 10 to 60 nucleotides (the fundamental unit of DNA and RNA) long, however most of the oligonucleotides synthesized are 11 nucleotides INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

17. 17 LEADING AND LAGGING STRANDS In E. coli , DNA polymerase III is the main polymerase involved in the replication process. There are two DNA molecules hard on work on each of the replication forks to synthesized new daughter strand. DNA polymerization can only proceed form 5’-3’ direction only. DNA double helix is antiparallel, one strand runs from 5’-3’ while other runs from 3’-5’ direction. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

18. 18 LEADING AND LAGGING STRANDS The helicase unzips the double-stranded DNA for replication, making a forked structure. The primase generates short strands of RNA that bind to the single-stranded DNA to initiate DNA synthesis by the DNA polymerase. This enzyme can work only in the 5' to 3' direction, so it replicates the leading strand continuously. Lagging-strand replication is discontinuous, with short Okazaki fragments being formed and later linked together. The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

19. 19 MAINTENANCE AND CLEAN UP CREW There are few more enzymes other than discussed previously which are needed for smooth replication process. A DNA clamp , also known as a sliding clamp or β- clamp, is a protein complex that protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. Topoisomerases also plays an important maintenance role in DNA replication. They participate in the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork. Finally, the RNA primers are removed by DNA polymerase I . The nicks that remain after the primers are replaced get sealed by the enzyme DNA ligase . INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

20. 20 DNA REPLICATION PROCESS INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

21. 21 DNA REPLICATION TERMINATION At the last stage of termination, two replication fork meets at terminator recognizing sequences, called as a Ter . It contains ten sequences, TerA to TerJ , which are binding sites for '' tus '' , terminator utilization substance. The Ter sequences are ~ 20 bp long and contain the conserved sequence 5'-GTGTGTTGT-3'. Ter sequences with TUS protein create a complex which arrests the replication fork and prevent them to escape. At this complex, the process of replication is completed and all other proteins and enzymes leave this site. Only DNA topoisomerase II remains in action, it cuts both strands, dissociates Ter -TUS complex and two different circular DNA is generated.  INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

22. 22 SUMMARY Helicase unzips two DNA strands by disrupting hydrogen bonds between nitrogenous bases Single strand binding proteins bind to single strands to prevent rewinding of the DNA strands. Topoisomerase move ahead of the replication fork to ease supercoiling. Primase synthesizes RNA primers complementary to the DNA template strand. DNA polymerase III strands DNA chain extension process by adding nucleotides to 3’-end. DNA polymerase I removes RNA primers from lagging and leading strands. DNA ligase seals nicks between DNA fragments. Tus protein binds to ter sequences, hence stops helicase activity and the replication process is terminated. INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim