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CYTOGENETICS
Lesson
[TRANS] LESSON 10: DNA REPLICATION & REPAIR
WHAT IS DNA REPLICATION?
● Creation of a new strand of DNA from an
existing template strand
● Semi-conservative replication
○ Replicated strand is composed of:
■ Old (original) template
■ New strand
● DNA acts as a template for its own
replication. Because the nucleotide A will
successfully pair only with T, and G with C, each
strand of a DNA double helix—labeled here as
the S strand and its complementary Sʹ
strand—can serve as a template to specify the
sequence of nucleotides in its complementary
strand. In this way, both strands of a DNA
double helix can be copied with precision.
● “For a cell to survive and proliferate in a chaotic
environment, it must be able to accurately copy the vast quantity of genetic information carried in its DNA. This fundamental process, called DNA replication, must occur before a cell can divide to produce two genetically identical daughter cells. In addition to carrying out this painstaking task with stunning accu- racy and efficiency, a cell must also continuously monitor and repair its genetic material, as DNA is subjected to unavoidable damage by chemi- cals and radiation in the environment and by reactive
molecules that are generated inside the cell.”
STARTING DNA REPLICATION
● Replication origins
○ DNA sequences where replication starts ○ Where initiator proteins binds ● Replication forks
● “ A DNA double helix is opened at replication
origins. DNA sequences at replication origins
are recognized by initiator proteins (not shown),
which locally pull apart the two strands of the
double helix. The exposed single strands can
then serve as templates for copying the DNA.”
SPEED OF DNA REPLICATION
● Replication is bidirectional
● Numerous replication forks are present in animal cells ○ ~ 10,000 in humans ● Speed: ○ ~1,000 nucleotide pairs per second in bacteria ○ ~ 100 nucleotide pairs per second in humans
● ” The two replication forks formed at a replication
origin move away in opposite directions. (A) These drawings represent the same portion of a DNA molecule as it might appear at different times during replication. The orange lines represent the two parental DNA strands; the red lines represent the newly synthesized DNA strands. (B) An electron micrograph showing DNA replicating in an early fly embryo. The particles visible along the DNA are nucleosomes, structures made of DNA and the histone protein complexes around which the DNA is wrapped.”
PROTEINS INVOLVED IN DNA REPLICATION
DNA POLYMERASE III
● Adds nucleotides to the 3’ end of a growing DNA
strand ● Uses complementary base pairs to select nucleotide to be added ○ Deoxyribonucleoside triphosphate
● Creation of a new strand only occurs in a 5’-to-3’ direction
PRIMASE
● A specialized RNA (ribonucleic acid) polymerase
● Creates an RNA primer as a starting point for new DNA ● ~ 10 bp long
● ” RNA primers are synthesized by an RNA
polymerase called primase, which uses a DNA strand as a template. Like DNA polymerase,
primase synthesizes in the 5ʹ-to-3ʹ direction. Unlike
DNA polymerase, however, primase can start a new polynucleotide chain by joining together two nucleoside triphosphates without the need for a
base-paired 3ʹ end as a starting point. Primase uses
ribonucleoside triphosphate rather than deoxyribonucleoside triphosphate.”
DNA POLYMERASE I
● Also known as repair polymerase
● Removes RNA primers and replaces with DNA
● ” Multiple enzymes are required to synthesize the
lagging DNA strand. In eukaryotes, RNA primers are made at intervals of about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. These primers are extended by a replicative DNA polymerase to produce Okazaki fragments. The primers are subsequently removed by nucleases that recognize the RNA strand in an RNA–DNA hybrid helix and degrade it; this leaves gaps that are filled in by a repair DNA polymerase that can proofread as it fills in the gaps. The completed DNA fragments are finally joined together by an enzyme called DNA ligase, which catalyzes the formation of a phosphodiester bond between the
3 ʹ-hydroxyl end of one fragment and the 5ʹ-phosphate
end of the next, thus linking up the sugar–phosphate backbones. This nick-sealing reaction requires an input of energy in the form of ATP.”
DNA LIGASE
● Joins the 5’-phosphate end of one DNA fragment to
the adjacent 3’-hydroxyl end of the next
● ” DNA ligase joins together Okazaki fragments on
the lagging strand during DNA synthesis. The ligase enzyme uses a molecule of ATP to activate the
5 ʹ phosphate of one fragment (step 1) before forming
a new bond with the 3ʹ hydroxyl of the other fragment
(step 2).”
DNA HELICASE
● Uses ATP hydrolysis to move forward and unzip
double stranded template DNA
● ”Two types of replication proteins—DNA helicases
and single-strand DNA-binding proteins—cooperate to carry out this task. A helicase sits at the very front of the replication machine, where it uses the energy of ATP hydrolysis to propel itself forward, prying apart the double helix as it speeds along the DNA.”
SINGLE-STRAND DNA-BINDING PROTEINS
● Attach to the unzipped template DNA to prevent
re-forming ● Keeps template elongated
● ”Single-strand DNA-binding proteins then latch onto
the single-stranded DNA exposed by the helicase, preventing the strands from re-forming base pairs and
SELF-CORRECTING DNA POLYMERASE
● Error rate: one error: 10^7 nucleotide pairs copied
● Ways to avoid errors: ○ Monitoring base pairing ■ Bases must be complementary before the reaction is catalyzed ○ Proofreading ■ Previous base pair is checked before adding a new base pair ■ Mistakes are corrected ■ Only occurs in the 5’-to-3’ direction
- During DNA synthesis, DNA polymerase proofreads its own work. If an incorrect nucleotide is accidentally added to a growing strand, the DNA polymerase cleaves it from the strand and replaces it with the correct nucleotide before continuing.
TELOMERES AND TELOMERASES
TELOMERES
● Repeating sequences found at the end of a
chromosome
● ” Without a special mechanism to replicate the
ends of linear chromosomes, DNA would be lost during each round of cell division. DNA synthesis begins at origins of replication and continues until the replication machinery reaches the ends of the chromosome. The leading strand is synthesized in its entirety. But the ends of the lagging strand can’t be completed, because once the final RNA primer has been removed, there is no mechanism for replacing it with DNA. Complete replication of the lagging strand requires a special mechanism to keep the chromosome ends from shrinking with each cell division.”
TELOMERASES
● Act as primers for the end of the lagging strand
● Replenish the telomeres
● ” Telomeres and telomerase prevent linear
eukaryotic chromosomes from shortening with each cell division. To complete the replication of the lagging strand at the ends of a chromosome, the template strand (orange) is first extended beyond the DNA that is to be copied. To achieve this, the enzyme telomerase adds to the telomere repeat sequences at
the 3ʹ end of the template strand, which then allows
the newly synthesized lagging strand (red ) to be lengthened by DNA polymerase, as shown. The telomerase enzyme itself carries a short piece of RNA (blue) with a sequence that is complementary to the DNA repeat sequence; this RNA acts as the template for telomere DNA synthesis. After the lagging- strand replication is complete, a short stretch of single-stranded DNA remains at the ends of the chromosome; however, the newly synthesized lagging strand, at this point, contains all the information presen in the original DNA.”
CAUSES OF DNA DAMAGE
DEPURINATION
● Random loss of A and G bases
● ” Depurination and deamination are the most
frequent chemical reactions known to create serious DNA damage in cells. (A) Depurination can remove guanine (or adenine) from DNA.”
DEAMINATION
● Loss of an amino group from a cytosine (C) in DNA to
produce uracil (U)
● ”(B) The major type of deamination reaction converts
cytosine to uracil, which, as we have seen, is not
normally found in DNA. However, deamination can occur on other bases as well. Both depurination and deamination take place on double-helical DNA, and neither break the phosphodiester backbone.”
ULTRA-VIOLET (UV) RADIATION
● Promotes covalent linkage between two adjacent
pyrimidine (C and T) bases
● ” The ultraviolet radiation in sunlight can cause the
formation of thymine dimers. Two adjacent thymine bases have become covalently attached to each other to form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damage.”
CAUSES OF DNA DAMAGE
SUBSTITUTION
● Nucleotide is replaced by another
● Produces mismatch
DELETION
● Removal of one or more nucleotides
MISMATCHES
● An incorrect pairing with one of the base pairs
● Results in permanent mutation in the next round of DNA replication
● ” Errors made during DNA replication must be
corrected to avoid mutations. If uncorrected, a mismatch will lead to a permanent mutation in one of the two DNA molecules produced during the next round of DNA replication.”
MISMATCH REPAIRS
● When a mismatch is detected, repair is done based
on information on the complementary strand ● Nucleases – degrades mismatched nucleotides ● Repair DNA polymerase – fills in missing nucleotides ● Ligase – reconnects the DNA backbones
● ” Mismatch repair eliminates replication errors and
restores the original DNA sequence. When mistakes occur during DNA replication, the repair machinery must replace the incorrect nucleotide on the newly synthesized strand, using the original parent strand as its template. This mechanism eliminates the error, and allows the original sequence to be copied during subsequent rounds of replication.”
damage) using an undamaged homologous double helix as a template. ● Highly accurate DNA replication and DNA repair processes play a key role in protecting us from the uncontrolled growth of somatic cells known as cancer.
