Editing Primer (molecular biology) (section)

== RNA primers ''in vivo'' ==
{{Further|DNA polymerase|DNA replication}}RNA primers are used by living organisms in the [[Initiation of DNA replication|initiation]] of [[DNA synthesis|synthesizing]] a strand of [[DNA]]. A class of enzymes called [[primase]]s add a complementary RNA primer to the reading template ''[[De novo synthesis|de novo]]'' on both the [[Leading strand|leading]] and [[lagging strand]]s. Starting from the free 3’-OH of the primer, known as the primer terminus, a DNA polymerase can extend a newly synthesized strand. The [[leading strand]] in DNA replication is [[DNA synthesis|synthesized]] in one continuous piece moving with the [[replication fork]], requiring only an initial RNA primer to begin synthesis. In the lagging strand, the template DNA runs in the [[Directionality (molecular biology)|5′→3′ direction]]. Since [[DNA polymerase]] cannot add bases in the 3′→5′ direction complementary to the template strand, DNA is synthesized ‘backward’ in short fragments moving away from the replication fork, known as [[Okazaki fragments]]. Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers. Along the DNA template, [[primase]] intersperses RNA primers that DNA polymerase uses to synthesize DNA from in the 5′→3′ direction.<ref name=":0" />

Another example of primers being used to enable DNA synthesis is [[Reverse transcriptase|reverse transcription]]. Reverse transcriptase is an enzyme that uses a template strand of RNA to synthesize a complementary strand of DNA. The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis.<ref name=":0" />

===Primer removal===
After the insertion of [[Okazaki fragments]], the RNA primers are removed (the mechanism of removal differs between [[prokaryote]]s and [[eukaryote]]s) and replaced with new [[deoxyribonucleotides]] that fill the gaps where the RNA  primer was present. [[DNA ligase]] then joins the fragmented strands together, completing the synthesis of the lagging strand.<ref name=":0" />

In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches the previous RNA primer. Then the enzyme simultaneously acts as a [[Phosphodiesterase I|5′→3′ exonuclease]], removing primer [[ribonucleotide]]s in front and adding [[deoxyribonucleotides]] behind. Both the activities of polymerization and excision of the RNA primer occur in the [[Upstream and downstream (DNA)|5′→3′]] direction,  and polymerase I can do these activities simultaneously; this is known as “Nick Translation”.<ref>{{Cite book |last=Doudna; Cox; O'Donnell |first=Jennifer; Michael M.; Michael |title=Molecular Biology: Principles and practice |publisher=W. H. Freeman |date=December 21, 2016 |isbn=9781319116378 |language=English}}</ref> Nick translation refers to the synchronized activity of polymerase I in removing the RNA primer and adding [[deoxyribonucleotides]]. Later, a gap between the strands is formed called a nick, which is sealed using a [[DNA ligase]].

In eukaryotes the removal of RNA primers in the [[lagging strand]] is essential for the completion of replication. Thus, as the lagging strand being synthesized by [[DNA polymerase δ]] in [[Upstream and downstream (DNA)|5′→3′]] direction, [[Okazaki fragments]] are formed, which are discontinuous strands of DNA. Then, when the DNA polymerase reaches to the 5’ end of the RNA primer from the previous Okazaki fragment, it displaces the 5′ end of the primer into a single-stranded RNA flap which is removed by nuclease cleavage. Cleavage of the RNA flaps involves three methods of primer removal.<ref name=":1">{{Cite journal |last1=Uhler |first1=Jay P. |last2=Falkenberg |first2=Maria |date=2015-10-01 |title=Primer removal during mammalian mitochondrial DNA replication |journal=DNA Repair |language=en |volume=34 |pages=28–38 |doi=10.1016/j.dnarep.2015.07.003 |pmid=26303841 |issn=1568-7864|doi-access=free }}</ref> The first possibility of primer removal is by creating a short flap that is directly removed by [[flap structure-specific endonuclease 1]] (FEN-1), which cleaves the 5’ overhanging flap. This method is known as the short flap pathway of RNA primer removal.<ref name=":2">{{Cite journal |last1=Balakrishnan |first1=Lata |last2=Bambara |first2=Robert A. |date=2013-02-01 |title=Okazaki fragment metabolism |journal=Cold Spring Harbor Perspectives in Biology |volume=5 |issue=2 |pages=a010173 |doi=10.1101/cshperspect.a010173 |issn=1943-0264 |pmc=3552508 |pmid=23378587}}</ref> The second way to cleave a RNA primer is by degrading the RNA strand using a [[RNase]], in eukaryotes it’s known as the RNase H2. This enzyme degrades most of the annealed RNA primer, except the nucleotides close to the 5’ end of the primer. Thus, the remaining nucleotides are displayed into a flap that is cleaved off using FEN-1. The last possible method of removing RNA primer is known as the long flap pathway.<ref name=":2" /> In this pathway several enzymes are recruited to elongate the RNA primer and then cleave it off. The flaps are elongated by a 5’ to 3’ [[helicase]], known as [[PIF1 5'-to-3' DNA helicase|Pif1]]. After the addition of nucleotides to the flap by Pif1, the long flap is stabilized by the [[replication protein A]] (RPA). The RPA-bound DNA inhibits the activity or recruitment of FEN1, as a result another nuclease must be recruited to cleave the flap.<ref name=":1" /> This second nuclease is [[Okazaki fragments#Dna2 endonuclease|DNA2 nuclease]], which has a helicase-nuclease activity, that cleaves the long flap of RNA primer, which then leaves behind a couple of nucleotides that are cleaved by FEN1. At the end, when all the RNA primers have been removed, nicks form between the [[Okazaki fragments]] that are filled-in with [[deoxyribonucleotides]] using an enzyme known as [[Ligase|ligase1]], through a process called [[Ligation (molecular biology)|ligation]].