Lecture 3- DNA Replication PDF

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Professor Kenworthy. These notes cover the DNA Replication of genetics....

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Lecture 3: DNA Replication 01/15/19 There are 3 major pathways of information flow in the cell: 1. Replication – information passes from one DNA to another DNA 2. Transcription- information passes from DNA to RNA 3. Translation- information passes from RNA to protein

DNA Replication  The process of copying one DNA molecule to another DNA molecule  Necessary for cellular division and critical for the transmission of genetic information  Has to be accurate, has the ability to correct mistakes, and has to be fast  Each strand is a template for a new strand o Hydrogen bonds break and the two DNA strands separate o Each strand acts as a template for the synthesis of new DNA strands o New strands = daughter strands o Template strands = parent strands DNA replication is semiconservative process. - Results in exact duplicates of the original double stranded DNA molecule - New dsDNA (double strand molecule) – one original strand and one new strand Slide 5 blue are original and red are newly produced DNA (daughter double stranded molecule). One template and one daughter double stranded molecule

*Meselson – Stahl experiment - proved that DNA replicated semiconservitavely  DNA in E. Coli was labeled with 15N o Both strands were 15N (15N/15N)  Bacteria transferred to 14N media and allowed to proceed through one round of relication o One strand is 14N and the other is 15N (15N/14N)  Second round of replication o Both 14N/14N DNA and 15N/14N DNA present  Subsequent replications o Both present, but higher proportion of 14N/14N will be present Experiment: which model of DNA replication applies to E. Coli?

Conclusion: DNA replication in E. Coli is semiconservative. Process for semiconservative: Starts at 15/15 then transfers to 14 and replicates to 14/15 and two 15/15 then replicates again to have 14/14 as well as 14/15 and two 15/15 after the second round of replication.

Origin of Replication  DNA synthesis begins at an origin of replications o Prokaaryotes: oriC o Eukaryotes: origin recognition complex (ORC) DNA replication continued: o Mediated by large enzymes called DNA polymerase o Enzymes that synthesizes DNA DNA Polymerases: - Are primer depended - Primers- short DNA or RNA molecules with free 3’ OH group - Add new nucleotides one at a time to the 3’ end of a primer - Matches a new nucleotides based on the template strand (A/T, G/C) *DNA elongation always always always proceeds in the 5’ to 3’ direction (the daughter strand produced this way)

Replication Fork - DNA synthesis must take place simultaneously but in opposite direction on the two template strands. - 5’ primer laid down then connected to it is 3’ okazaki fragments attach as short segments in lagging strands in the opposite direction of the bubble. Leading strand has one primer in the direction of the bubble

DNA Synthesis  Synthesis of the leading strand proceeds in the same direction as the replication fork o Synthesized continuously o Requires only one primer  Synthesis of the lagging strand must occur in the opposite direction of the moment of the replication fork o Requires multiple RNA primers  Laid down in intervals as the replication for moves o DNA is synthesized in short fragments – Okazaki fragments o The RNA primers must be replaced by DNA polymerase

Bacterial DNA Replication Bacterial chromosomes are circular 2 possible mechanisms: 1. Theta replication 2. Rolling circle replication Rolling circle replications: the F factor for E. Coli (How plasmids replicate) Replication begins by a break in one of the nucleotide strands. Does not need a primer. Begins at the 3’ end of the broken strand and the inner strand is used as a template. The 5’ end of the broken strand is displaced. Cleavage releases a single stranded linear DNA and a double stranded circular DNA.

Bacterial Replication Mechanisms 1. Initiations a. Single replication origin (oriC) b. Imitators proteins (DnaA) bind to oriC and cause a short segment of DNA to unwind 2. Unwinding a. Helicase (DnaB) binds to the short stretch of single stranded DNA i. DNA helicase – enzymes that break hydrogn bonds between the two strands of DNA ii. Binds to the lagging-strand template and moves 5’ to 3’ ( moves the replication fork) b. Single stranded binding (SSBs ) protein attach and stabilize the single stranded DNA during replication 3. Elongation a. Primers i. Elongation or adding of nucleotides requires a 3’ OH group ii. Primase synthesizes a short RNA primer and places it at the origin iii. Leading strand to one primer iv. Lagging strand to primer at the beginning of each Okazaki fragment b. DNA polymerase elongates the polynucleotide strand c. DNA polymerase – 5 types i. Polymerase 3 is the major replication enzyme DNA Polymerase 1 replaces all RNA primer used to initiate elongation.

DNA polymerase 1 can’t facilitate binding to the 5’ end of the DNA inserted by DNA polymerase 3. So a nick remains after DNA polymerase 3 where 5’ end is. DNA polymerase 3 - The enzymes that catalyze the synthesis of DNA by using a template strand  Synthesis occurs in the 5’ to 3’ direction - Proceeds along a single stranded molecule of DNA  Free nucleotides are attached to complement the existing base using hydrogen bonds  The new nucleotide is attached to the previous nucleotides Replication mechanisms cont:  The DNA polymerase also have nuclease activity o The ability to break phosphodiester bonds in the sugar phosphaye backbone of a nucleic acid chain, (Cuts DNA) o Provides a built in mechanism for correcting rare errors o If DNA polymerase 3 adds an incorrect nucleotide it can cut out the wrong nucleotides o Two types of nucleases  Exonucleases – all have exonuclease activity in the 3’ to 5’ direction.  Endonucleases  These reduce the frequency of mutation

DNA polymerase and DNA ligase 1. 2. 3. 4. 5. 6.

DNA pol 1 binds to a single stranded gap between DNA and RNA primer Pol 1 removes an RNA prime nucleotide Fills the gap with a DNA nucleotide Pol 1 removes each RNA primer nucleotide Replaces it with DNA nucleotide When primer removal is done, DNA ligase replaces pol 1 at DNA-RNA single stranded gaps 7. Catalyzes formation of phosphodiester bond to join Okazaki fragment. DNA ligase seals the gap between the two DNA polymerase. Bacterial Replication mechanism cont: 4. Termination a. Occur when two replication forks meet

b. In some cases specific termination sequcnes block further replications

Eukaryotic Replication Differnces between Eukaryotic and Prokaryotic - Multiple orgins of replication - Slower rate of DNA synthesis - Telomere replication Eukaryotic Replication Mehcanisms o Replications occur in a fixed location  Polymerase does not move along the DNA strand Eukaryotic Repliaction mechanisms 1. Initiation a. at each origin a multiprotein of origin of recognition (ORC) binds to intiate unwinding i. timing regulated so that each segemtn is replicated only once per cell cycle b. polymerase interact with the Proliferating Cell Nuclear Anitgen (PNCA) i. functions as a sliding clamp to help bind polymerases 2. Unwinding and Elongation a. Fuctions similar to the bacterial replication but differ by protein used Look at table 7.2 slide 31. Universal terms for prokaryotic and eurkaryotic cells except polymerase Eukaryotic DNA polymerases 1. Alpha (Primase activity) laying RNA primer 2. Delta (laggings strand) 3. Epsilon (leading strand synthesis) Termination:  Unlike bacterial chromosomes which are circular Eukaryotes are linear o Ends are known as telomeres  Problems with completing replications  Gap left by removal of the last RNA primer o DNA polymerase can’t fill the gap  Suggests that chromosomes would become shorter through time > chromones destabilization> cell death.

 Telomeres are a short repeated sequence/stretch of nucleotides. EX: 5’ – TTGGGG-3’  Telomerase enzyme – lengthens the ends of chromosomes and extends the template strand telomere so the Alpha polymerase can add an RNA primer so the chromosome end can be replicated.  Telomerase is only present in specific cells such as germ cells or early embryonic cells....