Cell Biology DNA Replication Repair and Recombination Alberts

DNAstrand can serve as a template Preferential binding occurs between pairs of nucleotides (A with T, and G with C) that can form base pairs. This enables each strand to act as a template forming its complementary strand.

DNAacts as a template for its own duplication Both S strand its complementary S’ strand – can serve as a template to specify the sequence of nucleotides in its complementary strand. In this way, double-helical DNA can be copied precisely. The copy must be carried out with speed and accuracy: in about 8 hours, a dividing animal cell will copy the equivalent of 1000 books like ECB and get no more that a letter or two wrong. This feat is performed by a cluster of proteins that form a replication machine.

Each of the two strands of DNAis used as a template for the formation of a complementary DNA strand Semiconservative replication: • Each parental strand serves as the template for one new strand; • Each daughter DNA double helix is composed of one of the original (old) strands plus one strand that is completely new.

ADNAdouble helix is opened at its replication origin Replication initiator proteins recognize sequences of DNA at replication origins and locally pry apart the two strands of the double helix. The exposed single strands can then serve as templates for copying the DNA.

Replication forks move away in opposite directions from multiple replication origins in a eucaryotic chromosome The particles visible along the DNA are nucleosomes in the early embryo of a fly in an electron micrograph. (1), (2) and (3) represent the same portion of a DNA at successive stages of replication. The orange lines represent the parental DNA strands; the red lines represent the newly synthesized DNA.

DNAis synthesized in the 5’-to-3’ direction Addition of a deoxyribonucleotide to the 3’-hydroxyl end of a polynucleotide chain is the fundamental reaction by which DNA is synthesized. The new DNA chain is thereby synthesized in the 5’-to-3’ direction. The nucleotides enter the reaction as nucleoside triphosphates. Base paring between the incoming deoxyribonucleotide and the template strand guides the formation of a new strand of DNA that is complementary in nucleotide sequence to the template chain. The DNA polymerase catalyzes the addition of nucleotides to the free 3’ hydroxyl on the growing DNA strand. Breakage of a phosphoanhydride bond in the incoming nucleoside triphosphate releases a large amount of free energy and thus provide the energy for the polymerization reaction.

DNAreplication forks are asymmetrical Because both the new strands are synthesized in the 5’-to 3’ direction, the lagging strand of DNA must be made initially as a series of short DNA strands called Okazaki fragments that later joined together. The DNA strand that is synthesized discontinuously in this way is called the lagging strand; the other strand, which is synthesized continuously, is called the leading strand.

DNApolymerase proofreads its own work If an incorrect nucleotide is added to a growing strand, the DNA polymerase will cleave it from the strand replace it with the correct nucleotide before continuing. First, the DNA polymerase carefully monitors the baseparing between each incoming nucleotide and the template strand. Only when the match is correct does DNA polymerase catalyze the nucleotide addition reaction. Second, when DNA polymerase makes a rare mistake and add the wrong nucleotide, it can correct the error through an activity called proofreading.

DNApolymerase contains separate sites for DNA synthesis and proofreading DNA polymerase is in the polymerizing mode (left) and proofreading mode (right). The catalytic sites for the polymerization activity (P) and error-correcting proofreading activity (E) are indicated. When an incorrect nucleotide is added, the newly synthesized DNA (red) transiently unpairs from the template (orange), and the polymerase undergoes a conformational change that moves the error-correcting catalytic site into a position where it can remove the most recently added nucleotide.

Onthe lagging strand, DNAis synthesized in fragments DNA polymerase cannot start a completely new DNA strand. In eucaryotes, RNA primers are made at intervals of about 200 nucleotides on the lagging strand by primase (an RNA polymerase), and each RNA primer is about 10 nucleotides long. Primers are removed by nucleases that recognize an RNA strand in an RNA/DNA helix and degrade it; this leaves gaps that are filled in by a DNA repair polymerase that can proofread as it fills in the gaps. The completed fragments are finally joined together by DNA ligase, which catalyzes the formation of a phosphodiester bond between the 3’-OH end of one fragment and the 5’phosphate end of the next, thus linking up the sugar-phosphate backbones. Thus nick-sealing reaction requires an input of energy in the form of ATP or NADH.

DNAsynthesis is carried out by a group of proteins that act together as replication machine One DNA polymerase is on the leading strand the other polymerase on the lagging strand. Both are held on to the DNA by a circular protein clamp that allows the polymerase to slide. At the head of the fork, a DNA helicase uses the energy of ATP hydrolysis to propel itself forward and thereby separate the strands of the parental DNA double helix. Singlestrand DNAbinding proteins maintain these separated strands as singlestranded DNA to provide access for the primase and polymerase.

DNAsynthesis is carried out by a group of proteins that act together as replication machine A current view of how the replication proteins are arranged at the replication fork when the fork is moving. The DNA on the lagging strand is folded to bring the lagging-strand DNA polymerase molecule in contact with the leading-strand DNA polymerase molecule This folding also bring the 3’ end of each completed Okazaki fragment close to the start site for the next Okazaki fragment. Therefore, The lagging strand DNA polymerase can be reused to synthesize successive Okazaki fragments.

Telomeres allow the completion of DNAsynthesis at the ends of eucaryotic chromosomes To synthesize the lagging strand at the very end of a eucaryotic chromosome, the DNA replication machinery required a length of template DNA extending beyond the DNA that is to be copied. The telomerase adds a series of DNA repeats to the 3’ end of the template strand, which then allows the lagging strand to be completed by DNA polymerase. In humans, the nucleotide sequence of the repeat is GGGGTTA. The telomerase enzyme carries a short piece of RNA whose sequence is complementary to the DNA repeat sequence; this RNA acts as the template for the telomerase DNA synthesis.

Errors made during DNAreplication must be corrected to avoid mutation (A) If uncorrected, the mismatch will leads to a permanent mutation in one of the two DNA molecules produced by the next round of DNA replication. (B) If the mismatch is ‘repaired’ using the newly synthesized DNA strand as the template, both DNA molecules produced by the next round of DNA replication will contain a mutation. (C) If the mismatch is corrected using the original template (old) strand as the template, the mutation is eliminated.

DNAmismatch repair proteins correct errors that occur during DNA replication In eucaryotes, newly replicated DNA strands are preferentially nicked, it is these nicks (single-stranded breaks) that appear to provide the signal that directs the mismatch repair machinery to the appropriate strand. A DNA mismatch distorts the geometry of the DNA. The distortion is subsequently recognized by the DNA mismatch repair proteins, which then remove the newly synthesized DNA. The gap in the newly synthesized DNA is replaced by a DNA polymerase that proofread as it synthesizes and is sealed by DNA ligase. Such nicks are known to occur in the lagging strands and are observed also occur, although less frequently, in the leading strands.

Depurination and deamination are the most frequent chemical reactions known to create serious DNAdamage in cells During the time it takes to read this sentence, a total of about a trillion (1012) purine bases (A and G) will be lost from DNA in the cells of your body by a spontaneous reaction called depurination. Another reaction is the loss of an amino group (deamination) from cytosine in DNA to produce the base uracil.

The ultraviolet radiation in sunlight causes DNA damage Two adjacent thymine bases have become covalently attached to one another to form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damage.

Chemical modifications of nucleotides, if left unrepaired, produce mutations (A) Deamination of cytosoine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. Deamination of cytosine produces uracil which differs from cytosine in its base-paring properties and preferentially base-pairs with adenine. The DNA replication machinery therefore inserts an adenine when it encounters a uracil on the template strand. (B) Depurination, if uncorrected, can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it can skip to the next complete nucleotide, thus producing a nucleotide deletion in the newly synthesized strand. In other cases, an incorrect nucleotide was placed across from the missing base.

The basic mechanism of DNArepair involves three steps: excision, resynthesis, and ligation Step 1 (excision), the damage is cut out by one of a series of nucleases, each specialized for a type of DNA damage. Step 2 (resynthesis), the original DNA sequence is restored by a repair DNA polymerase, which fills in the gap created by the excision events. Step 3 (ligation), DNA ligase seals the nick left in the sugar-phosphate backbone of the repaired strand. Nick sealing requires energy from ATP hydrolysis to remakes the broken phosphodiester bond between the adjacent nucleotides Some types of DNA damage (the deamination of cytosine nucleotide) involve the replacement of a single nucleotide. For the repair of other kinds of DNA damage, such as thymine dimer, a longer stretch of 10 -20 nucleotides is removed from the damaged strand.

Cells can use nonhomologous end-joining to repair double-strand breaks In human somatic cells, the most common means of repairing double-strand breaks is by a mechanism called nonhomologous end-joining. In this process, the two broken ends are simply brought together by a specialized group of enzymes and rejoined by DNA ligation. This “quick and dirty’ mechanism alters the original DNA sequence during the process of repairs The alterations are usually short deletions.

Homologous recombination : Repair of DNAdouble-strand breaks Homologous recombination is often initiated when a double-strand break occurs shortly after a stretch of DNA is replicated as the duplicated DNA helices are still in close proximity (A). A nuclease generates singlestranded ends at the break by chewing back one of the complementary DNA strands (B). With the help of specialized enzymes, one of these single strands then invades the homologous DNA duplex by forming base pairs with its complementary strand (C). If this sampling results in extensive base pairing, a branch point is created where the two DNA strands – one from each duplex - cross (C). The invading strand is elongated by a repair DNA polymerase, using complementary strand as a template (D).

Homologous recombination : Repair of DNAdouble-strand breaks The branch point then ‘migrates’ as the base pairs holding together the duplexes break, and new ones form (E). Repair is completed by additional DNA synthesis, followed by DNA ligation (F). The net result is two intact DNA helices, where the genetic information from one was used as a template to repair the other. Homologous recombination can also be used to repair many other types of DNA damage. The ‘all-purpose’ nature of recombinational repair probably explains why this mechanism has been conserved in virtually all cells on earth.