ADN Replication

Chapter 11 : DNA Replication Outline: * Semiconservative Replication o Meselson-Stahl Experiment * DNA polymerases and DNA elongation * Molecular model of DNA replication o Initiation of

Chapter 11 : DNA Replication

Outline:

* Semiconservative Replication

o Meselson-Stahl Experiment

* DNA polymerases and DNA elongation

* Molecular model of DNA replication

o Initiation of Replication

o Semidiscontinuous DNA replication

o Rolling circle replication

* Replication of telomeres in eukaryotes

DNA replication underlies the process of inheritance at all levels (cellular, organismal, population) DNA replication occurs as prelude to cell division ( S phase of cell cycle in eukaryotes) DNA in all organisms is the end point in a continuous series of replications going back to the origin of life, almost 4 billion yrs ago DNA replication is based on complementarity of DNA molecules and on ability of proteins to form specific interactions with specific sequences of DNA

o Conservative model: parental strands rejoin after they are used as templates, resulting

in two DNA moleucles, one made of two parental strands, and the other made entirely of newly synthesized DNA

o Dispersive model: parental DNA cleaved into DNA segments that act as templates for the synthesis of new DNA and then somehow segments reassemble into double stranded DNA made of parental and progeny DNA which are interspersed

o All three models made different predictions about the nature of DNA after one and two rounds of replication (Fig 11.1)

seperated from 14N DNA by ultracentifugation in a CsCl gradient.

o 15N-labeled bacteria were then transferred to medium containing 14N and allowed to grow for several generations, and sampled after each replication cycle

o After one generation in 14N, all the DNA had a density intermediate from 15N-DNA and14N-DNA, just as predicted by the semiconservative and dispersive models

+ this result ruled out the conservative model because it predicted that there should be two bands (one containing light DNA and the other heavy DNA)

o To distinguish between the semiconservative model and the dispersive model, E.coli were grown for another generation Two bands were observed, as expected by the semiconservative model The dispersive model predicted that there should only be one band, therefore it was also ruled out The results were all consistent with the

semiconservative model

o

* Semiconservative DNA replication also occurs in eukaryotes (see harlequin

chromosomes in Fig 11.3)

DNA polymerases and DNA elongation

* In 1955, Arthur Korberg identified the first DNA polymerase (DNA Pol I) Initially it wasthought to be the main DNA replication enzyme, but mutant E.coli defective in the gene encoding for DNA pol I divided normally, indicating that there must be other enzymes involved

* Five DNA polymerases have now been identified in E coli DNA Pol II, IV, and V are involved in DNA repair DNA pol I and III are involved in DNA replication

* All DNA polymerases catalyze the polymerization of nucleotide precursors (dNTPs) into

a DNA chain The reaction is shown in Fig 11.4 and has three main features:

1 DNA pols catalyze the formation of a phosphodiester bond between the 3'-OH group ofthe deoxyribose on the last nucleotide in the chain and the 5'-phosphate of the incoming nucleotide The energy is supplied by the hydrolysis of the two phosphates from the dNTP All DNA polymerases require a primer (i.e they can not add the first nucleotide )

2 DNA polymerases require a template The particular nucleotide added depends on correct complementary base pairing with the template DNA pols are fast In E coli, DNA pol I and II can polymerize ~ 850 nt per sec In humans, its a lot slower (60-90 nt/sec)

3 All DNA polymerases synthesize DNA in the 5' to 3' direction

* DNA pol I and II also have exonuclease activity

o DNA pol I and III have 3'-> 5' exonuclease activity This is a proofreading mechanism DNA pols add an incorrect base with a frequency of 10-6 When an incorrect base is added, the enzyme detects that it made a mistake, and uses its 3' to 5' exonuclease activity to move back and remove the incorrect base With proofreading, the error rate drops to 10-9

o DNA pol I also has 5' -> 3' exonuclease activity This allows it to remove DNA or RNA from the 5' end of a moleecule This is essential during DNA replication of the lagging strand

Model of DNA Replication in E coli

* The bare-bone mechanics of DNA replication is similar in all organisms However, we will only focus on DNA replication in E coli, where it is best understood Along the way, significant differences between prokaryotic and eukaryotic DNA replication will be

highlighted

* Basic research into the mechanisms of DNA replication in E coli (as well as

transcription and translation) has led to the identification and cloning of dozens of genes involved in these processes (Table 11.1) The creative use of these gene products has given us a tremendous power to manipulate genes and genomes according to our will

Initiation of Replication

* Initiation of replication starts at a DNA sequence called the replicator, which includes the origin of replication (OriC) (AT-rich) where DNA is denatured into single strands to

form a replication bubble At either end of a bubble there is a replication fork, where DNAsynthesis occurs, using each separated strand as a template.

o Circular genomes of prokaryotes contain a single origin of replication

o In eukaryotes, linear chromosomes contain many origins of replication (allows faster replication)

o Synthesis proceeds bidirectionaly at replication fork Eventually, replicated double helices join each other, producing two daughter molecules (Fig 11.9)(sister chromatids,

in eukaryotes)

* Initiation of replication starts with the binding of an initiator protein which denatures the oriC and then recruits a DNA helicase (one for each strand) which untwists the DNA

in both directions (energy comes from hydrolysis of ATP) (Fig 11.5)

* Next, each helicase recruits a DNA primase to form a primosome DNA primase makes the necessary RNA primers ( 5-10 nts) needed by DNA polymerase III

* The next step involves the assembly of the rest of the proteins involved in DNA

replication These proteins associate to form a replisome There is a replisome at each

replication fork

Semidiscontinuous DNA replication

* The replication steps are identical at each replication fork, so we focus on just one Theentire process is shown in Fig 11.6

* After the helicase unwinds the DNA, the single stranded DNA is prevented from

reannealing by binding to single-strand DNA-binding proteins (SSBs) (about 200 /rep fork)

* DNA pol III dimer (part of replisome) now initiates polymerization by adding dNTPs to the RNA primer on each of the strands Because strands in double helix are in antiparallelconfiguration, and DNA polymerases add dNTPs in 5' to 3' direction, the two strands are synthesized differently:

o Leading strand synthesized continuously; only one primer required; DNA pol III moves

in same direction as replication fork

o Lagging strand synthesized discontinuously as Okazaki fragments, which are later ligated by DNA ligase Each Okazaki fragment requires a primer DNA pol III moves in

opposite direction to replication fork.

* In Lagging-strand synthesis, DNA Pol III ends polymerization when it encounters

double stranded DNA ahead (from previous Okazaki fragment) It dissociates from the

DNA, leaving a gap in one strand This gap is recognized as damaged DNA and is

repaired by DNA Pol I

* DNA Pol I removes primers and fills in gaps (has 5'-3' exonuclease activity)

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* DNA ligase joins 3' end of one Okazaki fragment to 5' end of downstream Okazaki

fragment (Fig 11.7)

* As helicase unwinds DNA ahead of replication fork, positive supercoils form elsewhere

in the molecule For replication fork to move, the helix must rotate (estimated at 50

revolutions/sec) The problem of supercoiling is solved by the action of topoisomerases (specifically a Gyrase) which introduce negative supercoils to counteract positive

supercoils intoduce by helicases

Rolling circle replication

* For many viral DNAs and some plasmids (e.g F plasmid in E coli), rolling circle

replication has been demonstrated

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* Synthesis usually continues beyond a single chromosomal unit This results in many head-to-tail copies of the plasmid, which is then cut and rejoined into new circular

molecules

Replication of telomeres in eukaryotes

* There are special problems associated with replication of the ends of linear

chromosomes (called telomeres) Recall that DNA polymerases only add nucleotides to the 3' end of a growing chain When the linear chromosomes of eukaryotes replicate, the resulting daughter molecules will each have an RNA primer left over at the 5'end (Fig 11.14) This RNA primer is removed, leaving a single stranded DNA segment If not fixed,this single-stranded DNA region will get degraded, and the linear chromosomes will get shorter with each round of DNA replication

* In most eukaryotes, an enzyme called telomerase, maintains the ends of chromosomes

by adding telomere repeats to chromosome ends The mechanism is shown below (and inFig 11.5)

* Telomerase is a ribonucleoprotein (has RNA molecule as part of its structure) which adds tandem repeats to the 3' end of chromosomes using an RNA molecule as a

template After is has added many telomeric repeats and has left, a new DNA molecule ismade starting from a new RNA primer, which is again is removed, but by this time the chromosme has already been extended

* The absence of telomerase activity in cells is correlated with senescence of cells (i.e die after certain number of cell divisions) Conversely, enhanced telomerase activity correlated with cell immortalily (i.e cells divide indefinately)

o cells with short telomerse undergo fewer doublings than ones with long telomerase

o fibroblasts form individuals with progeria (rare disease characterized by premature aging) have short telomeres

o most somatic cells have no active telomerase (divide only 20-60 times)

o sperm cells, stem cells and unicellular eukaryotes (essentially immortal ) have active telomerase and stable telomeres

o cancer cells, which are also essentially immortal, have active telomerase (promising target for drug design)

o Elimination of telomerase activity in somatic cells may be a cellular senescence

mechanism that protects multicellular organisms from cancer

o RNA processing in eukaryotes

Genes and RNA

Biological information flow from DNA to protein requires an RNA intermediate RNA is produced by a process that copies the nucleotide sequence in DNA to produce a transcript This process is called transcription

Properties of RNA

1 Single stranded, but can undergo intramolecular base-pairing

- forms variety of 3D structures specified by sequence

2 Ribose sugar (not deoxyribose)

3 Uracyl in place of thymine

Classes of RNA

* There are a variety of different RNAs that can be classified into two classes

o 1 Informational RNAs (e.g messenger RNA)

+ intermediate which is later translated into protein

+ most genes encode mRNA

o 2 Functional RNAs

+ never translated

+ diverse roles in cell

+ main classes of functional RNAs play critical roles in various steps in the information processing of DNA to protein:

# rRNA - components of ribosome

# tRNA - bring amino acids to mRNA during translation

# snRNA (small nucleolar RNAs) - involved in splicing of introns

# scRNAs (small cytoplasmic RNAs) - protein trafficking

* All DNA and RNA function is based on two key elements:

o 1 Complementary bases in single stranded nucleotide chains can H-bond to form double stranded structures

o 2 Specific sequences can be recognized by specific nucleic-acid binding proteins

Making functional transcripts

* Transcription uses one DNA strand as template

o Strands of double helix must be separated, so that one of these strands (template strand) can serve as template to direct the synthesis of transcript

* Either strand along the chromosome can serve as template, but for a given gene, its always the same strand

* RNA polymerase catalyzes the synthesis of RNA using DNA template (Fig 13.1)

o RNA grows in 5' to 3' direction, and the template is read in the 3' to 5' direction

o sequence of RNA is complementary to template strand (noncoding strand), but the same as nontemplate strand (coding strand) except T replaced with U

* A typical prokaryotic gene has the folowing features:

RNA Polymerases

* Prokaryotes have only one RNA Polymerase but eukaryotes have 3:

1 RNA Pol I: transcribes rRNA genes

2 RNA Pol II: transcribes protein encoding genes

3 RNA Pol III: transcribes other functional RNAs (tRNAs, snoRNAs etc )

* In eukaryotes, transription takes place in nucleus

* In prokaryotes, transcription and translation are coupled

* Transcription involves 3 distinct stages: initiation, elongation, and termination

Initiation

* In E coli, transcription requires a complex of RNA polymerase and the sigma factor (s) which binds to a promoter The RNA polymerase core enzyme (4 has four subunits, two

a, one b and one b') complexed with the sigma factor is known as the holoenzyme Once transcription is initiated, the sigma factor dissociates

* promoter = DNA sequence to which RNA Pol binds to initiate transcription

o note that by convention, gene is labelled the same way as RNA transcript So promoter

is at 5' end of gene (Fig 13.3)

* RNA pol + sigma factor scans DNA for promoter sequence, binds DNA at the promoter sequence (- 10 region and -35 region), unwinds it, and begins synthesis of a transcript attranscription initiation site Promoter sequences are not transcribed NOTE: RNA pol doesnot need a primer to initiate RNA synthesis not does it need a helicase

o there are consensus sequences for all promoters in E coli A consensus sequence is thesequence found most frequently at each position E.g consensus sequence at -10 position

* What is described above is the minimum required for transcription initiation In chapter

19 we will study how genes are regulated in prokaryotes in more detail

+ Rho is a protein that binds RNA terminator sequence and then uses energy from ATP hydrolysis to separate transcript from RNA polymerase.

+ Rho-dependent terminators lack hairpin loop

* The transcript that is made is called mRNA and in prokaryotes it has the following

structure (note that it's always larger than what is needed to encode the polypeptide):

* Prokaryotes have coupled transcription and translation, so that even as mRNA is being transcribed, ribosomes attach to 5' end and begin translation

* Many mRNAs in prokaryotes are polycistronic, i.e they can encode more than one

polypeptide (E.g lac operon)

RNA processing in Eukaryotes

* Transcription in eukaryotes is more complicated, in that there are more regulatory

sequences involved, and there is a sequential assembly of many different transcription

factors at the promoter before RNA polymerase binds and initiates trancription We will study transcription in eukaryotes in more detail in chapter 20

* In this chapter, we will focus on processing of the initial transcript

* In eukaryotes, the initial product of transcription (primary transcript) is processed in

several ways before transport to cytosol In prokaryotes, there is no such processing

* Processing performed by RNA binding proteins

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* Processing involves (Fig 13.9 and Fig 13.11):

o 1 addition of 5' cap

+ Guanyltransferase adds 7-methylguanosine using a 5'-to-5' triphosphate linkage

+ protects transcript aginst degradation by exonucleases

+ also important for binding of ribosome during translation

o 2 addition poly(A+) tail

+ transcript cleaved 20 bases downstream of AAUAAA sequence near 3' end by an endonuclease, then 50-250 adenine nucleotides added by Poly(A) polymerase

+ Poly(A+) tail required for efficient transport out of the nucleus into cytoplasm Once in cytoplasm, polyA tail also protects against early degradation by exonulceases

o 3 RNA splicing to remove introns (Fig 13.13)

+ the GU-AG rule

+ spliceosome

Chapter 10: DNA as a genetic material

Search for genetic material

* Scientists reasoned early on that whatever the genetic material turned out to be, it had

to have 3 important characteristics:

1 Store information (about structure, function, development, reproduction)

2 Replicate accurately (so progeny can receive information from parents)

3 Capable of change Without mutation there is no variation and adaptation Evolution does not occur

Griffith’s transformation experiment[/b]

* In 1928, Frederick Griffith was working with two different strains of Streptococcus pneumoniae (causes pneumonia)

1 S strain: forms smooth colonies; highly infectious because it forms a capsule which allows bacteria to evade immune system of host (virulent)

2 R strain: forms rough colonies; harmless because it lacks a capsule, therefore gets detected by immune system early and effectively (avirulent)

* Griffith injected mice with the different strains and checked for virulence The

experiment was as follows:

* The experiment showed that bacteria need to be alive and to have a polysaccharide capsule to be infectious and kill the host More importantly, it also showed that bacteria could uptake genetic material from their surroundings Griffith called this material the transforming principle, which he believed was protein The real importance of Griffith’s experiment is that it provided the experimental basis for further experiments on the chemical nature of the transforming principle

Transformation experiments of Avery, MacLeod and McCarty

* In the 1930’s and 40’s these researchers followed up on Griffiths experiments, by fractionating the heat killed cell extract into proteins, and nucleic acids Only the nucleic acid fraction was capable of causing transformation of a rough strain into a virulent S strain This indicated that proteins were not the transforming principle, and so could not

be the genetic material