Okazaki fragments are short DNA nucleotide sequences with an RNA primer at the 5′ end.
They are synthesized discontinuously during DNA replication.
These fragments are later joined together by the enzyme DNA ligase.
Okazaki fragments contribute to the formation of the lagging strand in DNA replication.
Reiji Okazaki and his wife, Tsuneko Okazaki, discovered Okazaki fragments in 1968.
Their discovery occurred while they were researching the replication of bacteriophage DNA in Escherichia coli.
In bacteria and bacteriophage T4, Okazaki fragments range in length from 1000 to 2000 nucleotides.
In eukaryotes, Okazaki fragments are approximately 150 to 200 nucleotides long.
Discovery of Okazaki fragments
In 1963, Tsuneko and Reiji Okazaki began studying DNA replication at Nagoya University, Japan.
They concluded that semiconservative replication of DNA could be explained if daughter strands were produced in vivo by a discontinuous mechanism.
They investigated the replication point to determine whether the lagging strand, which develops in the 3’–5′ direction, could be synthesized as short fragments in the 5’–3′ direction.
If true, linking these short pieces would allow the daughter strand to elongate.
To verify discontinuous strand replication, Reiji Okazaki conducted a pulse-chase experiment.
Actively replicating DNA was first exposed to “hot” tritiated nucleotides for a 5-second pulse, allowing radioactive nucleotides to integrate into DNA strands.
The DNA was then isolated by chasing the pulse for varying durations using “cold” unlabeled nucleotides.
After centrifugation, the DNA was analyzed for radioactivity.
With short chase durations of 7 to 15 seconds, most radioactivity was found in small fragments higher in the centrifuge tube.
With longer chases, more radioactivity appeared in larger, lower strands.
This experiment demonstrated that small fragments form on the lagging strand during synthesis and are later joined into longer strands.
These short DNA fragments were named “Okazaki fragments” by Rollin Hotchkiss in 1968 at the Cold Spring Harbor Symposium on the Replication of DNA in Micro-organisms.
Formation of Okazaki fragments
The DNA replication fork is formed when the double helix is unwound, and DNA helicase splits the complementary strands during replication.
DNA primase and DNA polymerase begin acting at the replication fork to create a new complementary strand.
The two unwound template strands are replicated differently due to enzymes’ limitation to work only in the 5′ to 3′ direction.
The leading strand’s template runs 3′ to 5′, allowing DNA polymerase to continuously synthesize the new strand by following the replication fork.
The lagging strand’s template runs 5′ to 3′, forcing DNA polymerase to move away from the replication fork, resulting in discontinuous synthesis.
This discontinuous synthesis leads to periodic breaks and the formation of Okazaki fragments.
RNA primers made by primase serve as starting points for synthesizing each Okazaki fragment.
DNA polymerase δ in eukaryotes and DNA polymerase I in prokaryotes extend the RNA primer toward the replication fork.
Since primase and polymerase move opposite to the fork’s direction, they must repeatedly stop and restart as helicase continues unwinding DNA.
Each Okazaki fragment requires a new RNA primer to begin synthesis.
The priming process occurs in three steps:
PriA protein displaces SSB proteins from a short DNA segment.
Primase (DnaG) binds to PriA.
Primase synthesizes a short RNA primer of 11–12 bases.
DNA polymerase releases the DNA when a new Okazaki fragment starts forming and relocates to begin a new strand from the 3′ end of a new RNA primer.
The clamp-loading complex disassembles and repositions the sliding clamp.
Only one sliding clamp, used by the lagging strand’s polymerase, is frequently released and reattached to a new site.
This is because only the lagging strand requires repeated clamp removal and reloading.
Enzymes with endonucleolytic activity later remove the RNA primers, including:
Ribonuclease H (RNAse H)
Flap endonucleases (FENs)
Dna2 helicase/nucleases
In eukaryotes, FENs are separate enzymes, whereas in prokaryotes, the FEN nuclease is a domain of DNA polymerase I.
The exact mechanism for removing RNA-DNA primers from Okazaki fragments remains unclear.
DNA ligase joins Okazaki fragments into a continuous strand using phosphodiester linkages.
Because one strand is synthesized continuously and the other discontinuously, DNA replication is semi-discontinuous.
Significance of Okazaki fragments
Proper processing of Okazaki fragments is essential for accurate DNA replication and normal cell growth.
Defects in Okazaki fragment processing can lead to breaks in DNA strands.
Such defects may also cause various chromosomal abnormalities.
Chromosome mutations resulting from faulty Okazaki fragment processing can affect chromosome structure, the number of chromosome sets, or the number of individual chromosomes.
References
Balakrishnan, L., & Bambara, R. A. (2013). Okazaki fragment metabolism. Cold Spring Harbor Perspectives in Biology, 5(2), a010173. https://doi.org/10.1101/cshperspect.a010173
Clark, D. P., Pazdernik, N. J., & McGehee, M. R. (2019). Chapter 10 – Cell Division and DNA Replication. In Molecular Biology (3rd ed., pp. 296–331). Academic Cell. ISBN 9780128132883. https://doi.org/10.1016/B978-0-12-813288-3.00010-0
MacNeill, S. A. (2001). DNA replication: partners in the Okazaki two-step. Current Biology, 11(20), R842–R844. https://doi.org/10.1016/s0960-9822(01)00500-0
Okazaki, T. (2017). Days weaving the lagging strand synthesis of DNA – A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism. Proceedings of the Japan Academy, Series B, 93(5), 322–338. https://doi.org/10.2183/pjab.93.020
ITbM Nagoya University. Professors Tsuneko and Reiji Okazaki and the Okazaki Fragment. Accessed from: https://www.itbm.nagoya-u.ac.jp/Okazaki6/Okazaki_fragment.html
Reha-Krantz, L. J. (2013). Okazaki Fragment. In Brenner’s Encyclopedia of Genetics (2nd ed., pp. 158–160). Academic Press. ISBN 9780080961569. https://doi.org/10.1016/B978-0-12-374984-0.01087-1
Verma, P. S., & Agarwal, V. K. (2005). Replication of DNA. In Cell Biology, Genetics, Molecular Biology, Evolution and Ecology (Multicolor ed., p. 31). S. Chand & Company Ltd., Ram Nagar, New Delhi. ISBN 81-219-2442-1
