DNA Replication and Repair. Fundamental Properties of Cells. Introduction. All cells undergo DNA replication and cell division in order to give rise to a new generation of cells.
DNA Replication and Repair Fundamental Properties of Cells
Introduction • All cells undergo DNA replication and cell division in order to give rise to a new generation of cells Mitosis- Division of the nucleus of a eukaryotic cell into two daughter nuclei with identical sets of chromosomes
Cytokinesis • Division of cell cytoplasm and organelles of a cell into two daughter cells • It is important that each daughter cell has an exact copy of the parent cell’s DNA
DNA Replication • 1958- Matthew Meselsonand Frank Stahl devised a clever experiment that suggested that DNA replication is semiconservative • They grew E. coli in a medium using ammonium ions (NH4+) as the source of nitrogen for DNA (as well as protein) synthesis • They worked with two isotopes of nitrogen • 14N is the common isotope of nitrogen, but they also used ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N.
Semiconservative Replication • Semiconservative replication is the process of replication in which each DNA molecule is composed of one parent strand and one newly synthesized strand (daughter strand) • Each daughter molecule receives one strand from the parent molecule plus one newly synthesized strand
The Process of DNA Replication • Replication begins when proteins bind at a specific site on the DNA known as the replication origin • In prokaryotes, the closed circular DNA has only one origin of replication • In eukaryotes, the linear DNA has multiple origins of replication • In both organisms, the two strands forming the DNA molecule cannot simply be pulled apart. Why?
Replication Origin • The two strands of the DNA molecule cannot be simply pulled apart because they are held together by hydrogen bonds that are twisted around each other to form a double helix.
DNA Helicase • To expose a template strand, the two parent DNA strands must be unravelledand kept separate. • DNA Helicaseis a specific enzyme that unwinds the double helix by breaking the hydrogen bonds between the base pairs
SSBs • DNA base pairs are complementary to each other and have a natural tendency to anneal • Anneal: the pairing of complementary strands of DNA through hydrogen bonding • Single-stranded binding proteins (SSBs) are proteins that keep separated strands of DNA apart after DNA helicase has unwound them SSBs bind to the exposed DNA single strands and block hydrogen bonding
DNA Gyrase • The bacterial enzyme that relieves any tension brought about by the unwinding of the DNA strands during replication • Gyrase works by cutting both strands of DNA, allowing them to swivel around one another, and then releasing the cut strands • Enzymes from the same family as gyrase have similar functions in eukaryotes
DNA Replication • DNA cannot be fully unwound because of its large size compared with the size of the cell • The diameter of a human cell is ~ 5 μm • The length of DNA is ~1 cm, • That is a 2000-fold difference • As a region of the DNA unwinds, replication begins in two directions from that origin
DNA Replication • New complementary strands are built as soon as an area of the DNA has been unwound • Replication fork: As the two strands of DNA are disrupted, the junction where they are still joined is called the replication fork
Replication Bubble • In eukaryotes, more than one replication fork may exist on a DNA molecule at once because of the multiple sites of origin • This results in hundreds or replication forks across a DNA strand and • allows for rapid replication of DNA • When two replication forks are quite near each other, a replication bubble forms
Origin of Replication Replication Bubble Replication Fork Parental Strand Daughter Strand
Origin of Replication Replication fork Eventually, the replication bubbles become continuous and the two new double-stranded daughter molecules are completely formed Replication bubbles
Building the Complementary Strands • In Prokaryotes, DNA Polymerase I, II, and III are the three enzymes that function in DNA replication and repair • In Eukaryotes, five different types of DNA Polymerase are at work
Review: Nucleic Acid • contains a chain of nucleotides • covalently linked together via • phosphodiester bonds • to form a sugar-phosphate backbone • with protruding nitrogenous bases.
Nucleotide • A nucleotide consists of • a nitrogenous base, • a sugar, and • a phosphate group
Nucleoside • A nucleoside consists of a nitrogenous base covalently attached • to a sugar (ribose or deoxyribose) • but without the phosphate group • Therefore, a nucleotide is a “nucleoside-mono-phosphate”
DNA Polymerase III • DNA polyermerase III is the primary enzyme responsible for replication. • It's main function is to add the 5' phosphate of a new nucleotide to and existing 3'-OH group. • Therefore, it synthesizes DNA in the 5' to 3'direction, i.e., it adds free deoxyribonucleosidetriphosphatesto a 3' end of an elongating strand
DNA Polymerase III • Why does it add deoxyribonucleosidetriphosphates? • The high energy of the triphosphatesis used to form bonds between the nucleotides. • The two outermost phosphates are liberated, leaving the innermost group still attached. • Once the two outermost phosphates have been released, the nucleoside triphosphate has become a nucleotide
DNA Polymerase III • This enzyme has its own limitations • It can't begin a new daughter strand by itself - it requires that there already be a 3'-OH end to add the next nucleotide to • Since DNA Polymerase III cannot initiate a new complementary DNA by itself, an RNA primer (10-60 base pairs of DNA) is annealed to the template strand
Primase • The enzyme that builds RNA primers in a 5' to 3' direction since DNA polymerase III cannot begin initiating on its own • This primer provides the free 3'-OH needed by DNA polymerase III. • This initiation sequence is temporary and is later removed and replaced by DNA • Once in place, DNA Polymerase III can start elongation by adding free to the complementary strand
Steps of DNA Replication • The first major step for the DNA Replicationis the breaking of hydrogen bonds between bases of the two antiparallel strands. • The unwounding of the two strands is the starting point. • Helicase is the enzyme that splits the two strands.
Steps of DNA Replication • The initiation point where the splitting starts is called "origin of replication" • The structure that is created is known as Replication Fork
nucleotides. Steps of DNA Replication • One of the most important steps of DNA Replicationis the binding of RNA Primasein the initiation point of the 3'-5' parent chain • RNA Primasecan attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases.
Steps of DNA Replication • When DNA is being replicated, two types of daughter strand are being produced • Leading strand • Lagging strand
DNA Leading Strand • Since DNA is always synthesized in the 5' to 3' direction and the template strands run antiparallel, only one strand is able to be built continuously, that is the Leading strand. The 3'-5' proceeding leading strand uses a 5'-3‘ template because DNA Polymerase can "read" the template and continuously adds nucleotides
DNA Lagging Strand • The other strand is synthesized discontinuously in short fragments in the opposite direction to the replication fork and is known as the Lagging strand The 5'-3‘ lagging strand uses a 3'-5‘ template because DNA Polymerase cannot "read" the template and continuously adds nucleotides. In the lagging strand the RNA Primase adds more RNA Primers.
etc). Okazaki Fragments • Short fragments of DNA that are a result of the synthesis of the lagging strand during DNA replication
DNA Polymerase III • The enzyme that adds free deoxyribonucleotidesfrom primer to primer forming Okazaki fragments
DNA Polymerase I • The enzyme that removes RNA primers from the leading and lagging strands and replaces the RNA primers with appropriate deoxyribonucleotides
DNA Ligase • The enzyme that joins the Okazaki fragments into one strand by creation of a phosphodiester bond • As the two strands of DNA are synthesized, two double-stranded DNA molecules are produced that automatically twist into a helix