Chapter 5: DNA Replication, Repair, and Recombination

Goals Illustrate how structure of DNA affects its function Describe enzymes involved in replication Summarize their functions Explain how DNA is repaired and why it needs to be repaired

DNA Structure (A Review) DNA consists of two strands Each strand is a polymer of nucleotides Strand has orientation due to nucleotide structure: 5’ and 3’ ends The two strands are antiparallel

DNA Function (A Review) DNA function is information storage Sequence of strand stores info Genes are copied into RNA (transcription) “Control elements” regulate protein interactions with DNA DNA passed on to descendant cells Accurate copying Repair of any damage to avoid changes Accurate subdivision Stored info determines cell’s functions; therefore best if transmitted error-free Copied DNA split between cells- also important, probably not covered (much?)

Functions Determine Form Double strands of DNA allow “easy” replication Rules for obligate pairing Each strand acts as template for the other Multiple proteins involved Act in concert Act as complexes Recognize DNA by shape of bases Sequence of nucleotides is what must remain unchanged, so that new daughter cells get correct instructions on genes to express and when and how much (as well as sequence of genes themselves) Theme of series is involvement of multiple proteins in carrying out DNA function, including “self-replication”- they must act together in appropriate order and often do so as large clusters of proteins interacting together

Paired DNA Strands Nucleotides on inside with phosphate and deoxyribose on outside There are hydrogen bond donors and acceptors not involved in base pairing These as well as several of donor/acceptors involved in base pairing can interact with proteins in the major and minor grooves

Replication Overview Replication complex binds to replication “origin” Double-stranded DNA is “melted” Each strand is used as a template for DNA synthesis

Mechanism of DNA Replication General Features of DNA Replication Semiconservative Complementary Base Pairing DNA Replication Fork is Assymetrical Replication occurs in 5’ 3’ Direction

Semi-conservative Replication

Semi-conservative Replication

DNA Replication

DNA Replication

Tools of Replication Enzymes are the tools of replication: DNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each other Primase - Provides an RNA primer to start polymerization Ligase - Joins adjacent DNA strands together (fixes “nicks”)

More Tools of Replication Helicase - Unwinds the DNA and melts it Single Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicase Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule Telomerase - Finishes off the ends of DNA strands

In the Beginning… Problems due to structure DNA is double-stranded Template must be single-stranded Strands must be separated Separation is difficult due to structure Melting strands causes tension elsewhere If unrelieved tension can snap DNA strand

Topoisomerase (Gyrase) Relieves stress caused by melting DNA Cleaves DNA and spins around itself to unwind helix Type I cleaves one strand, type II cleaves both Reseals DNA strands after relaxation achieved Use example of two ropes twined around each other, held at one end; stick finger into hole of a loop then pull two apart at on end- tension increases and finger gets squeezed

DNA Replication DNA Helicase Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA Moves 1000 bp/sec 2 helicases: one on leading and one on lagging strand SSB proteins aid helicase by destabilizing unwound ss conformation

Single Strand Binding Protein Binds to DNA with no sequence preference Binds tighter to single strand than double Keeps separated strands from rejoining

DNA Replication SSB proteins help DNA helicase destabilizing ssDNA

Primase Creates a primer for DNA polymerase Template-dependent An RNA polymerase Active briefly at beginning of strand synthesis

DNA Replication DNA Primase uses rNTPs to synthesize short primers on lagging Strand Primers ~10 nucleotides long and spaced ~100-200 bp DNA repair system removes RNA primer; replaces it w/DNA DNA ligase joins Okazaki fragments

DNA Polymerase Enzyme that synthesizes a DNA strand Uses existing strand as template Requires a free “3’ end” to add new nucleotides Has several catalytic functions Several forms exist

Eukaryotic DNA Polymerases Enzyme Location Function Pol  (alpha) Nucleus DNA replication includes RNA primase activity, starts DNA strand Pol  (gamma) Nucleus DNA replication replaces Pol  to extend DNA strand, proofreads Pol  (epsilon) Nucleus DNA replication similar to Pol , shown to be required by yeast mutants Pol  (beta) Nucleus DNA repair Pol  (zeta) Nucleus DNA repair Pol  (gamma) Mitochondria DNA replication

Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme Correct nucleotide has greater affinity for moving polymerase than incorrect nucleotide Exonucleolytic proofreading of DNA polymerase DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base paired DNA polymerase has separate catalytic site that removes unpaired residues at terminus

Maintenance of DNA Sequences High Fidelity DNA Replication Error rate= 1 mistake/109 nucleotides Afforded by complementary base pairing and proof-reading capability of DNA polymerase

DNA Replication DNA Polymerase held to DNA by clamp regulatory protein Clamp protein releases DNA poly when runs into dsDN Forms ring around DNA helix Assembly of clamp around DNA requires ATP hydrolysis Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

DNA Replication Okazaki Fragments RNA that primed synthesis of 5’ end removed Gap filled by DNA repair enzymes Ligase links fragments together

Extension - Okazaki Fragments DNA Pol. 3’ 5’ RNA Primer Okazaki Fragment DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction. 3’ 5’ RNA Primer DNA Pol. RNA and DNA Fragments DNA Polymerase falls off leaving a nick. 3’ 5’ RNA Primer Ligase Nick The nick is removed when DNA ligase joins (ligates) the DNA fragments.

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Bacteria Single Ori Initiation or replication highly regulated Once initiated replication forks move at ~400-500 bp/sec Replicate 4.6 x 106 bp in ~40 minutes

DNA Replication Initiation and Completion of DNA Replication in Chromosomes DNA Replication Begins at Origins of Replication Positions at which DNA helix first opened In simple cells ori defined DNA sequence 100-200 bp Sequence attracts initiator proteins Typically rich in AT base pairs

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Eukaryotic Chromosomes Have Multiple Origins of Replication Relication forks travel at ~50 bp/sec Ea chromosome contains ~150 million base pairs Replication origins activate in clusters or replication units of 20-80 ori’s Individual ori’s spaced at intervals of 30,000-300,000 bp

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Mammalian DNA Sequences that Specify Initiation of Replication 1000’s bp in length Can function when placed in regions where chromo not too condensed Human ORC required for replication initiation also bind Cdc6 and Mcm proteins Binding sites for ORC proteins less specific

The Eukaryotic Problem of Telomere Replication RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

Different types of Nucleotide Polymerases DNA polymerase Uses a DNA template to synthesize a DNA strand 2) RNA polymerase Uses a DNA template to synthesize an RNA strand (= transcription) 3) Reverse transcriptase Uses an RNA template to synthesize a DNA strand Found in many viruses Telomerase is a specialized reverse transcriptase

Telomerase Two components of the human telomerase: the human RNA subunit(hTR) 5’-CUAACCCUAAC-3’ The human telomerase reverse transcriptase(hTERT)

Telomerase Function: Specialized reverse transcriptase Prevents “shortening ends problem” problem by adding telomeres to the end Copies only a small segment of RNA that it carries by itself Requires a 3’ end as a primer Synthesis proceeds in 5’ – 3’ direction Synthesizes one repeat then repositions itself When active provides cell immortality

Telomerase is composed of both RNA and protein

What are telomeres? Telomeres are… Repetitive DNA sequences at the ends of all human chromosomes They contain thousands of repeats of the six-nucleotide sequence, TTAGGG In humans there are 46 chromosomes and thus 92 telomeres (one at each end)

Telomeres Repeated G rich sequence on one strand in humans: (TTAGGG)n Repeats can be several thousand basepairs long. In humans, telomeric repeats average 5-15 kilobases Telomere specific proteins, eg. TRF1 & TRF2 bind to the repeat sequence and protect the ends Without these proteins, telomeres are acted upon by DNA repair pathways leading to chromosomal fusions

What do telomeres do? They protect the chromosomes. They separate one chromosome from another in the DNA sequence Without telomeres, the ends of the chromosomes would be "repaired", leading to chromosome fusion and massive genomic instability.

Telomere function, cont’. Telomeres are also thought to be the "clock" that regulates how many times an individual cell can divide. Telomeric sequences shorten each time the DNA replicates.

Telomerase Telomerase binds to the telomer and the internal RNA component aligns with the existing telomer repeats. 2. Telomerase synthesizes new repeats using its own RNA component as a template 3. Telomerase repositions itself on the chromosome and the RNA template hybridizes with the DNA once more.

 and Primase

Structure of Telomeres Elongated strand of telomere repeats are rich in guanine nucleotides 5’-TTTTGGGGTTTTGGGGTTTTGGGG-3’ They have the capacity to hydrogen bond to one another in the form of G-quartets. These create three-dimensional structures.

How Does Telomerase Work? Telomerase works by adding back telomeric DNA to the ends of chromosomes, thus compensating for the loss of telomeres that normally occurs as cells divide. Most normal cells do not have this enzyme and thus they lose telomeres with each division.

How Does Telomerase Work? In humans, telomerase is active in germ cells, in vitro immortalized cells, the vast majority of cancer cells and, possibly, in some stem cells.

How Does Telomerase Work? Research also shows that the counter that controls the wasting away of the telomere can be "turned on" and "turned off". The control button appears to be an enzyme called telomerase which can rejuvenate the telomere and allow the cell to divide endlessly. Most cells of the body contain telomerase but it is in the "off" position so that the cell is mortal and eventually dies.

How Does Telomerase Work? Some cells are immortal because their telomerase is switched on Examples of immortal cells: blood cells and cancer cells Cancer cells do not age because they produce telomerase, which keeps the telomere intact.

Telomerase and Cancer There is experimental evidence from hundreds of independent laboratories that telomerase activity is present in almost all human tumors but not in tissues adjacent to the tumors.

Telomerase and Cancer Thus, clinical telomerase research is currently focused on the development of methods for the accurate diagnosis of cancer and on novel anti-telomerase cancer therapeutics

Experimentation Many experiments have shown that there is a direct relationship between telomeres and aging, and that telomerase has the ability to prolong life and cell division.