Mary K. Campbell Shawn O. Farrell �������������������������� Chapter Ten Biosynthesis of Nucleic Acids: Replication Paul D. Adams • University of Arkansas 1 Replication of DNA • Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist in linear and circular forms in linear and circular forms • Difficult to generalize about all cases of DNA replication • We will study the replication of circular double- stranded DNA and then of linear double-stranded DNA DNA • most of the details we discuss were first investigated in prokaryotes, particularly E. coli 2
Flow of Genetic Information in the Cell • Mechanisms by which information is transferred in the cell is based on “Central Dogma” 3 Prokaryotic Replication • Challenges in duplication of circular double-stranded DNA • achievement of continuous unwinding and • achievement of continuous unwinding and separation of the two DNA strands • arotection of unwound portions from attack by nucleases that attack single-stranded DNA • synthesis of the DNA template from one 5’ -> 3’ strand and one 3’ -> 5’ strand strand and one 3’ -> 5’ strand • efficient protection from errors in replication 4
Prokaryotic Replication (Cont’d) • Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands strands using the original strands as templates • Semiconservative replication: Semiconservative replication: each daughter strand contains one template strand and one newly synthesized strand • Incorporation of isotopic label as sole nitrogen source ( 15 NH Cl) sole nitrogen source ( 15 NH 4 Cl) • Observed that 15 N-DNA has a higher density than 14 N-DNA, and the two can be separated by density-gradient ultracentrifugation 5 Evidence for Semiconservative Replication 6
Which Direction does Replication go? • DNA double helix unwinds at a specific point called an origin of replication origin of replication • Polynucleotide chains are synthesized in both • Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional bidirectional in most organisms • At each origin of replication, there are two replication replication forks forks , points at which new polynucleotide chains are formed • There is one origin of replication and two replication • There is one origin of replication and two replication forks in the circular DNA of prokaryotes • In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin 7 Bidirectional Replication 8
DNA Polymerase Reaction • The 3’-OH group at the end of the growing DNA chain acts as a nucleophile. • The phosphorus adjacent to the sugar is attacked, • The phosphorus adjacent to the sugar is attacked, and then added to the growing chain. 9 DNA Polymerase • DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’ direction of the template) • the leading strand leading strand is synthesized continuously in the 5’ -> 3’ direction toward the replication fork • the lagging strand • the lagging strand lagging strand is synthesized semidiscontinuously (Okazaki lagging strand is synthesized semidiscontinuously (Okazaki Okazaki Okazaki fragments) fragments) also in the 5’ -> 3’ direction, but away from the replication fork • lagging strand fragments are joined by the enzyme DNA ligase DNA ligase 10
Properties of DNA Polymerases • There are at least five types of DNA polymerase DNA polymerase (Pol) in E coli , three of which have been studied extensively 11 Function of DNA Polymerase • DNA polymerase function has the following requirements: • all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP dATP, dGTP, and dCTP • Mg 2+ • an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication • DNA-Pol I: repair and patching of DNA (remove and • DNA-Pol I: repair and patching of DNA (remove and fill up primers in lagging strand) • DNA-Pol III: responsible for the polymerization of the newly formed DNA strand • DNA-Pol II, IV, and V: proofreading and repair enzymes 12
DNA Polymerase III DNA Polymerase III ���������������������� ����������� ����������� ��������������������� ��������������������� � ����������������������� � ����������������������� ���������������������������� ���������������������������� ����� ����� ��� ��!���� ����� ����� ��� ��!���� ����� ��� ��!���� ����� ��� ��!���� � � � �������"���� �������"�������������� ������������#$������� ��#$������������ ����� ������ ������ � %�����������������" %�����������������" ' ����� � �����������������&���������������� �����������������&���������������� ' ����� � �������(���������������"�������"������������� �������(���������������"�������"������������� ��������������������� ��������������������� ��� ��������������������� ��������������������� ��� ��� ��������� ��� ��������� ��������� ��������� IT’S COMPLICATED!!! 13 14
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17 Supercoiling and Replication • DNA gyrase DNA gyrase (class II topoisomerase) catalyzes reaction involving relaxed circular DNA: circular DNA: • creates a nick in relaxed circular DNA • a slight unwinding at the point of the nick introduces supercoiling • the nick is resealed • The energy required for this process is supplied by the hydrolysis of ATP to ADP and P i 18
Replication with Supercoiled DNA • Replication of supercoiled circular DNA • DNA DNA gyrase gyrase has different role here. It has different role here. It introduces a nick in supercoiled DNA • a swivel point is created at the site of the nick • a swivel point is created at the site of the nick • the gyrase opens and reseals the swivel point in advance of the replication fork • the newly synthesized DNA automatically assumes the supercoiled form because it does not have the nick at the swivel point • helicase helicase , a helix-destabilizing protein, promotes unwinding by binding at the replication fork • single-stranded binding (SSB) protein stabilizes single-stranded regions by binding tightly to them 19 Primase Reaction • The primase reaction • RNA serves as a primer in DNA replication • primer activity first observed in-vivo . • Primase Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence • Synthesis and linking of new DNA strands • begun by DNA polymerase III • the newly formed DNA is linked to the 3’-OH of the • the newly formed DNA is linked to the 3’-OH of the RNA primer • as the replication fork moves away, the RNA primer is removed by DNA polymerase I 20
21 Replication Fork General Features 22
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