Genetics: DNA Replication

The relationship between structure and function is manifest in the double helix Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Fig. 16-7a 5 end Hydrogen bond 3 end 1 nm 3.4 nm Figure 16.7 The double helix 3 end 0.34 nm 5 end (a) Key features of DNA structure (b) Partial chemical structure

Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

Template or parental strands Daughter strands Fig. 16-9-3 Template or parental strands Daughter strands A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept

DNA replication is rather remarkable…. Replication of chromosomal DNA occurs very rapidly Very accurately at the appropriate time in the life of the cell

Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model They labeled the nucleotides of the old strands with a heavy isotope of nitrogen (N15), while any new nucleotides were labeled with a lighter isotope (N14).

Alternative methods of DNA replication Fig. 16-10 First replication Second replication Parent cell The two parent strands rejoin (a) Conservative model Alternative methods of DNA replication New strand contains one old and one new. (b) Semiconserva- tive model Figure 16.10 Three alternative models of DNA replication Each strand is a mix of old and new (c) Dispersive model

Density centrifugation using a CsCl gradient Fig. 16-11 EXPERIMENT 1 Bacteria cultured in medium containing 15N 2 Bacteria transferred to medium containing 14N RESULTS Density centrifugation using a CsCl gradient 3 DNA sample centrifuged after 20 min (after first application) 4 DNA sample centrifuged after 40 min (after second replication) Less dense More dense CONCLUSION First replication Second replication Conservative model Figure 16.11 Does DNA replication follow the conservative, semiconservative, or dispersive model? Semiconservative model Dispersive model

What Meselson and Stahl did not know was how replication took place? In 1960, Arthur Kornberg from Washington University, purified DNA polymerase from E.coli that was capable of synthesizing DNA. Today we know that more than a dozen enzymes and other proteins participate in DNA replication

Getting Started Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” Replication proceeds in both directions from each origin, until the entire molecule is copied. The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication.

DNA replication always adds nucleotides onto the 3’OH of the growing strand. DNA synthesis is always 5’ to 3’ on the template strand.

Regulation of DNA replication (prokaryotes) Specific proteins such as DnaA protein found in E.coli recognize DNA boxes within the origin (OriC in prok) and begin the replication.

Also a DAM protein methylates adenines to prompt regulation

In eukaryotes, much more complicated.

Difference in prokaryotic and eukaryotic DNA replication A eukaryotic chromosome usually has many origins of replication. The prokaryotic chromosome has one.

Parental (template) strand Fig. 16-12 Origin of replication Parental (template) strand Daughter (new) strand http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/animations/replication/replication.swf Replication fork Double- stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand Figure 16.12 Origins of replication in E. coli and eukaryotes 0.25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

Many enzymes are involved and are grouped together forming a DNA Replication Complex and the DNA winds through the complex like a reel in a movie projector.

At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding protein (SSB)binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

Single-strand binding proteins Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure 16.13 Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase

The workhorses…..DNA polymerases DNA polymerase is the enzyme(s) that catalyzes the formation of covalent bonds between adjacent nucleotides. DNA polymerases III adds new bases. DNA polymerase I removes RNA primers and fills in with DNA. DNA polymerases II, IV, and V help in repair and replication of damaged DNA.

DNA polymerase III Is a large molecule made of 10 different subunits; the complex is called DNA polymerase III holoenzyme.

The polymerases are like hands.

DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end. A short RNA primer is added initially. The 3 end of the RNA primer serves as the starting point for the new DNA strand. An enzyme called primase adds the RNA primer according to base-pairing. DNA polymerase I removes the RNA primers

RNA primers begin the process. Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure 16.13 Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase RNA primers begin the process.

Synthesizing a New DNA Strand How fast is this? The rate of elongation is about 750 nucleotides per second in bacteria and 50 per second in human cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate dNTP (base, sugar, 3 phosphates) As each nucleoside joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate. This supplies the energy for the process.

Nucleoside triphosphate

Nucleoside triphosphate Fig. 16-14 New strand 5 end Template strand 3 end 5 end 3 end This is a Source of Energy for DNA replication Sugar A T A T Base Phosphate C G C G G C G C DNA polymerase 3 end A T A T Figure 16.14 Incorporation of a nucleotide into a DNA strand 3 end C Pyrophosphate C Nucleoside triphosphate 5 end 5 end http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/animations/replication/replication.swf

Antiparallel Elongation The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication DNA polymerases add nucleotides only to the free 3end of a growing strand or to a primer; therefore, a new DNA strand can elongate only in the 5 to 3direction

Along one template strand of DNA, the DNA polymerase III synthesizes a leading strand continuously, moving toward the replication fork. A sliding-clamp protein moves DNA polymerase along the template strand.

Leading strands proceed toward the replication fork. Fig. 16-15a Leading strands proceed toward the replication fork. Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Figure 16.15 Synthesis of the leading strand during DNA replication

a NEW DNA strand can elongate only in the 5 to 3direction 5 Fig. 16-15b DNA can only add at the 3’ end of a growing DNA strand or a primer. Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 DNA pol III Parental DNA 3 5 Figure 16.15 Synthesis of the leading strand during DNA replication a NEW DNA strand can elongate only in the 5 to 3direction 5 3 5

To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments. Another DNA polymerase (I) replaces the RNA primer with DNA nucleotides. The fragments are joined together by DNA ligase.

Single-strand binding protein Overall directions of replication Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Helicase Leading strand 5 DNA pol III 3 3 Primer Primase 5 Parental DNA 3 Figure 16.17 A summary of bacterial DNA replication DNA pol III Lagging strand 5 DNA pol I DNA ligase 4 3 5 3 2 1 3 5 http://www.johnkyrk.com/DNAreplication.html

Two DNA polymerase III proteins act in concert to replicate the leading and lagging strands. The term dimeric DNA polymerase is used to describe the two DNA polymerase holoenzymes that move as a unit toward the replication fork. For this to occur, the lagging strand is looped out with respect to the DNA polymerase that synthesizes the lagging strand. This loop allows the laggings-strand polymerase to make DNA in a 5’ to 3’ direction yet move toward the opening of the replication fork.

The DNA Replication Complex Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules HHMI's BioInteractive - DNA replication (advanced detail) HHMI's BioInteractive - DNA replication (basic detail)

Fig. 16-UN5

DNA replication occurs with a high degree of fidelity. H bonding between matched pairs is more stable than mismatched pairs. The active site of DNA polymerase fits the correct base match with precision in an induced fit. Enzymatic removal of mismatched nucleotides. Proof- reading….DNA polymerase removes mismatched or damaged nucleotides by exonucleases and replaces them.

Repair at the 3’ end

Telomeres in eukaryotic replication Linear chromosomes contain telomeres at both ends Telomeric sequences consist of a moderately repetitive tandem Why are they needed? DNA polymerase is unable to replicate the 3’ ends of DNA strands so chromosomes would become progressively shorter. Telomerase prevents chromosome shortening by synthesizing additional repeats of telomeric sequences. Telomerase contains RNA and proteins. The RNA contains a complementary sequence to the DNA of the telomeric region and by a type of reverse transcription, replaces the DNA.

Can’t replicate here.

Unfortunately…. Telomerase is active only in germ cells, some types of stem cells such as embryonic stem cells, and certain white blood cells. And certain cancer cells

Summary of DNA replication in eukaryotic cells and prokaryotic cells Prok – one replication bubble; Euk – many Prok – circular chromosome; Euk- linear chromosomes with telomeres at ends Prok – a few types of DNA polymerase; Euk – many types of DNA polymerases Euk – more complicated, a lot we do not know about it for sure.

It looks like that the DNA was cut. It should have two bands, one is 1 It looks like that the DNA was cut. It should have two bands, one is 1.66kb and the other one is 4.1kb. There might be a weak band below or around the broken line of the gel on line 2 and 4 (it should be there). The uncut lines are correct (the supercoil DNA is the dominant form and run fast, the open circular is the weak band of slow running). I do not know why the ladder did not show up. We just used it and it was good.  2016